CN104982241B - A kind of method for evaluating transgenic pest-resistant rice aquatic environment security - Google Patents
A kind of method for evaluating transgenic pest-resistant rice aquatic environment security Download PDFInfo
- Publication number
- CN104982241B CN104982241B CN201510470544.9A CN201510470544A CN104982241B CN 104982241 B CN104982241 B CN 104982241B CN 201510470544 A CN201510470544 A CN 201510470544A CN 104982241 B CN104982241 B CN 104982241B
- Authority
- CN
- China
- Prior art keywords
- rice
- water
- transgenic
- field
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 121
- 235000009566 rice Nutrition 0.000 title claims abstract description 121
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 46
- 241000607479 Yersinia pestis Species 0.000 title claims abstract description 36
- 240000007594 Oryza sativa Species 0.000 title 1
- 241000209094 Oryza Species 0.000 claims abstract description 120
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 116
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 43
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 34
- 241000894007 species Species 0.000 claims abstract description 23
- 238000005070 sampling Methods 0.000 claims abstract description 20
- 238000000926 separation method Methods 0.000 claims abstract description 14
- 238000001556 precipitation Methods 0.000 claims abstract description 11
- 238000003973 irrigation Methods 0.000 claims description 16
- 230000002262 irrigation Effects 0.000 claims description 16
- 238000011144 upstream manufacturing Methods 0.000 claims description 15
- 239000002689 soil Substances 0.000 claims description 14
- 238000004458 analytical method Methods 0.000 claims description 11
- 241000238631 Hexapoda Species 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000011536 extraction buffer Substances 0.000 claims description 10
- 238000007710 freezing Methods 0.000 claims description 10
- 230000008014 freezing Effects 0.000 claims description 10
- 238000011835 investigation Methods 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 7
- 241000700141 Rotifera Species 0.000 claims description 7
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- 210000004700 fetal blood Anatomy 0.000 claims description 6
- 238000000751 protein extraction Methods 0.000 claims description 6
- 238000008157 ELISA kit Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 241000239250 Copepoda Species 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- FRTNIYVUDIHXPG-UHFFFAOYSA-N acetic acid;ethane-1,2-diamine Chemical class CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O.NCCN FRTNIYVUDIHXPG-UHFFFAOYSA-N 0.000 claims description 3
- 238000004820 blood count Methods 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 230000003287 optical effect Effects 0.000 claims description 3
- 239000000575 pesticide Substances 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 3
- 239000010865 sewage Substances 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000009333 weeding Methods 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 241001247197 Cephalocarida Species 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 239000002202 Polyethylene glycol Substances 0.000 claims 1
- 238000004108 freeze drying Methods 0.000 claims 1
- 239000008267 milk Substances 0.000 claims 1
- 210000004080 milk Anatomy 0.000 claims 1
- 235000013336 milk Nutrition 0.000 claims 1
- ZPIRTVJRHUMMOI-UHFFFAOYSA-N octoxybenzene Chemical compound CCCCCCCCOC1=CC=CC=C1 ZPIRTVJRHUMMOI-UHFFFAOYSA-N 0.000 claims 1
- 229920001223 polyethylene glycol Polymers 0.000 claims 1
- 238000001179 sorption measurement Methods 0.000 claims 1
- 238000011156 evaluation Methods 0.000 abstract description 12
- 238000007781 pre-processing Methods 0.000 abstract 1
- 238000007619 statistical method Methods 0.000 abstract 1
- 241000219146 Gossypium Species 0.000 description 14
- 229920000742 Cotton Polymers 0.000 description 13
- 230000007613 environmental effect Effects 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 6
- 244000037671 genetically modified crops Species 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 235000013305 food Nutrition 0.000 description 5
- 241000238421 Arthropoda Species 0.000 description 4
- 241001064674 Chrysopa septempunctata Species 0.000 description 4
- 241000238571 Cladocera Species 0.000 description 4
- 241001494246 Daphnia magna Species 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 108700019146 Transgenes Proteins 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000251468 Actinopterygii Species 0.000 description 3
- 208000003643 Callosities Diseases 0.000 description 3
- 241000131329 Carabidae Species 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 238000012502 risk assessment Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241000255967 Helicoverpa zea Species 0.000 description 2
- 241000143392 Oar Species 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 244000062645 predators Species 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 241000195628 Chlorophyta Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000005667 attractant Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000031902 chemoattractant activity Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 101150065438 cry1Ab gene Proteins 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 150000002171 ethylene diamines Chemical class 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 239000003621 irrigation water Substances 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 230000009571 larval growth Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000001926 trapping method Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Biodiversity & Conservation Biology (AREA)
- Botany (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- Environmental Sciences (AREA)
- Catching Or Destruction (AREA)
Abstract
The present invention provides a kind of method for evaluating transgenic pest-resistant rice aquatic environment security, comprises the following steps：Transgenic rice paddy and non-transgenic rice field arrangement and management；The measure of foreign protein in rice field water layer and mud, includes extracting and the measure of sample preprocessing and foreign protein；The separation of planktonic organism in the water layer of rice field, including water sampling, precipitation separation planktonic organism；The measure of plankton species and abundance in the water layer of rice field, including identification of living body sum gauge number；Safety evaluatio of the plantation of transgenic pest-resistant rice to planktonic organism, statistical analysis is carried out to plankton species, quantity, generation period using analysis software, draw the parameters such as diversity indices, evenness index, and be compared with conventional rice field, so as to evaluate transgenic pest-resistant rice aquatic environment security.The present invention has actual application value for the aquatic environment security of system evaluation transgenic pest-resistant rice.
Description
Technical field
The present invention relates to genetically modified crops technical field of ecological risk assessment, and in particular to one kind evaluates transgenic pest-resistant water
The method of rice aquatic environment security.
Background technology
Paddy rice is one of staple crops of China, and the resistance for strengthening paddy rice itself by transgenic breeding method is disease pest
Evil preventing and treating provides efficient solution route.Successfully having cultivated at present a collection of have high yield, Resistant and anti-removes
The Transgenic Rice of the characters such as careless agent, some of transgenic rice lines have come into the countries such as environmental release test
Bio-safety evaluation phase, technological reserve is provided for the Food Security of China.However, the plantation of genetically modified crops may
A series of safety problem can be brought, thus causes extensive concern and the arguement in the whole world.Because paddy rice is in the importance of China,
The concern of the department to transgenic paddy rice security such as environmental protection and agricultural seems to exceed any one other genetically modified crops, relevant to turn
The research of trans-genetic hybrid rice ecological risk assessment receives great attention.
In recent years, with regard to some transgenic pest-resistant rice strains gene assembly, the resistance risk of target pest, to non-target
The influence and toxalbumin of biological and Arthropod Communities in Rice Fields have been achieved for some progress in terms of the residual in soil,
Certain experience and technology is have accumulated for the safety evaluatio of transgenic paddy rice.Conventional research majority is only limited to examine or check transgenosis
Influence of the insect-proof rice to Paddy field athropods, and seldom to other important monoids in the Freshwater ecosystems of rice field --- it is aquatic
Protozoan (the important bait of fish) is kept a close eye on.Whether can be primary to water body dynamic for the large-scale plantation of transgenic paddy rice
Thing group brings adverse effect, there is great influence, and research report and the result accumulation of current this respect are very few.Therefore, commenting
During valency transgenic paddy rice Environmental security, it is necessary to supplement and strengthen the part to aquatic environment security implication.
The influence of transgenic crops on soil ecosystem is the focus paid close attention in recent years because soil be material and
The important place of energy circulation, foreign protein it is most advanced enter soil, in theory soil ecosystem most probable potentially born
Face rings.2000, U.S. EPA (US Environmental Protection Agency) gave birth to genetically modified crops to soil
The influence of state system is classified as the important component of risk assessment, and the influence of transgenic crops on soil ecosystem is after turning base
Another focus after being influenceed because of crop and nearly edge species gene stream, target pest resistance problem and on nontarget organism is asked
Topic.One recent studies on shows that large area plantation transgenic corns may be impacted to neighbouring aquatic ecosystem.The research
Proving the pollen and other parts of transgenic corns can flow into the irrigation canals and ditches around milpa, and disclose transgenosis jade with experiment
The accessory substance of rice can increase a kind of death rate of the aquatic insect referred to as the phryganeid, reduce its growth rate.
In many places of China, rice field and fish pond often weave in, and have rice field fish culture, rice-duck intergrowth etc.
Traditional Ecological cropping pattern, along with the special irrigation method of rice terrace, i.e., the series connection from upstream field to downstream field is irrigated.If
Be present the safety issue that there are similar transgenic corns to water ecosystem in transgenic paddy rice, then easily feed through to dirty
Paddy field and fishpond ecosystem.Therefore, whether transgenic paddy rice has safety issue more to paddy field and downstream aquatic ecosystem
Plus be worth noting.
It is related to method of the transgene cotton to Evaluation of Biocompatibility in Chinese patent CN104361251A, by setting up frequency
Ventilating type insecticidal lamp traps the adult of cotton field insect；Pass through the arthropod that the bodily form on naked eyes investigation whole cotton is larger；Using
Pest sucking device draws the less arthropod of all bodily forms in cotton complete stool and ground areas；Examined and inhaled using Stereo microscope
The arthropod that worm device is collected back is classified.Without reference to the content to aquatic zooplankter safety evaluatio.
Chinese patent CN104133054A provides the biologicall test side that a kind of evaluation transgene cotton influences on chrysopa septempunctata
Method, chrysopa septempunctata larva is fed with the transgene cotton blade cotton bollworm larvae of 1-3 days is taken food；Observe the development of chrysopa septempunctata larval growth
Situation, so as to evaluate influence of the transgene cotton to chrysopa septempunctata, the bioassay method is widely applicable, and to utilizing, " transgenosis is planted
Influence of the high dose albumen to predator can in thing-insect-predator " nutrition relationship evaluation genetically modified plants
Effectively determined.
Chinese patent CN102986462A is related to a kind of inspection that transgenic Bt cotton insect resistace is evaluated using the blue or green ground beetle of double spots
Survey method.This method utilizes the blue or green ground beetle-bollworm-cotton of food chain relation, i.e. pair spot in Ecosystem of Cotton Field, with predator
Killing extent of the quantity reflection transgenic Bt cotton of population to target organismses-lepidopterous insects (bollworm).Planted for many years
Turn in Bt cottons cotton field and control cotton cotton field, be combined the blue or green ground beetle of the double spots of trapping using trapping method and attractant method, compare two
The blue or green ground beetle quantity of double spots grown cotton in cotton field.Without reference to the content to aquatic zooplankter safety evaluatio.
Chinese patent CN102948399A is related to a kind of method for breeding of aquatile Daphnia magna.Overcome traditional Daphnia magna
The defect of toxicological experiment technology, establishes the method that a set of pure rice raises Daphnia magna, including：Nutrient solution, ground rice solution
Configuration, the raising and collection of young flea.The present invention raises Daphnia magna using pure rice, overcomes the medium-and-large-sized flea selectivity of conventional method
Absorb the defects of the natural food without taking food or seldom taking food rice such as green alga；And eliminate because green algae nutrient composition is uncertain
Easily the unfavorable factor of influence experimental result, provides reliable experiment for evaluation transgenic paddy rice security and ensures.The method is only
Suitable for genetically modified plants Environmental security Lab-evaluation.
Chinese patent CN102169117A is related to one kind and turns base based on fuzzy mathematics and Canonical correspondence analysis method to evaluate
The technical system influenceed by planting on soil micro-ecosystem and its application.Consider edaphon and thing there is provided one kind
The evaluation method of Physicochemical index, using this method can the plantation of effective evaluation genetically modified crops soil micro-ecosystem system brought
Influence.Without reference to the content to aquatic zooplankter safety evaluatio.
As seen from the above analysis, transgenic pest-resistant rice environmental safety assessment is only highlighted to the ground ecosystem
Influence, to geobiont and aquatile influence research it is less.In theory, the toxalbumin of transgenic pest-resistant rice can be with
By being secreted into soil and water body for root system, it can also enter bury by the residuum of the chip even aerial part of paddy rice
Earth, so as to produce influence to underground or aquatic tiny organism and meiofauna.Therefore, invention is needed badly a kind of suitable for aquatic life
The method of thing safety evaluation.
The content of the invention
It is an object of the invention to provide a kind of method for evaluating transgenic pest-resistant rice aquatic environment security, pass through design
The step such as the measure of foreign protein, the separation of planktonic organism and measure, can systematically be assessed in rice field arrangement, water layer and mud
The aquatic environment security of transgenic pest-resistant rice.
To achieve these goals, the technical solution adopted by the present invention is as follows：
A kind of method for evaluating transgenic pest-resistant rice aquatic environment security, including following process：
(1) transgenic rice paddy and non-transgenic rice field arrangement and management；
(2) in rice field water layer and mud foreign protein measure；
(3) in the water layer of rice field planktonic organism separation；
(4) in the water layer of rice field plankton species and abundance measure；
(5) safety evaluatio of the plantation of transgenic pest-resistant rice to planktonic organism.
According to above scheme, transgenic rice paddy and non-transgenic the rice field arrangement and management comprise the following steps：
1) physical features is smooth, 3 to 5 pieces of the convenient field in water source (3 to 5 repetitions i.e. for environmental safety assessment) for selection,
Field area 400-600m2, aspect ratio range 2: 1 to 1: 1, field periphery and water source are without Sewage Pollution；
2) by step 1) in field be divided into around cell, field build impermeable paddy field ridge and hardening irrigation canals and ditches；It is sudden and violent to prevent
Good gutter is set up on rain flood irrigation, field periphery；
3) in step 2) in upstream cell plantation transgenic pest-resistant rice, downstream cell plantation non-transgenic paddy rice, in
Between two pieces of non-transgenic cells irrigated with the water in field of upstream transgenosis cell, two pieces of cells of both sides are directly filled with the water in water source
Irrigate；
4) from transplanting to the maturity period, chemical pesticide is not applied, artificial weeding, mid-term not dry field.
According to above scheme, the concrete scheme that the field is divided into cell is：Upstream cell close to water source is set side by side
Two pieces of transgenosis cells are put, the downstream cell positioned at transgenosis cell downstream is set up in parallel 4 Kuai Fei pavilions gene cells；Described two pieces
Connection irrigation canals and ditches, downstream are set up between the upstream and water source of transgenosis cell respectively respectively with two pieces of middle non-transgenic cells to lead to
Cross irrigation canals and ditches connection；Two pieces of the outside of the non-transgenic cell is connected by irrigation canals and ditches with the water source respectively；The 4 Kuai Fei pavilions base
Because cell sets escape canal respectively.
According to above scheme, the measure of foreign protein in the rice field water layer and mud, including the pretreatment of sample and outer
The extracting of source protein specifically includes following steps with being determined by enzyme linked immunosorbent assay (ELISA method)：
1) water sampling and pretreatment：Water sampling cell includes all transgenosis cells and non-transgenic cell, uses
10ml centrifuge tubes carry out field water sampling, and each cell takes 5 pipes, take upper strata clear water, bring back interior, every part of sample is gone to respectively
In new 10ml centrifuge tubes, then freezed, sealed membrane is sealed up after freezing, hole is pricked on film with syringe needle, be then placed in freezing
Drying machine is concentrated to dryness；Dried sample is put into 4 DEG C of Cord bloods, it is to be measured in 24 hours；
2) mud sample collection and pretreatment：Soil sample collection cell includes all transgenosis cells and non-transgenic cell,
Respectively pull out 5 for root and stem of certain plants paddy rice and take rhizosphere mud, load 50ml centrifuge tubes, take back interior and be put into -20 DEG C of refrigerator freezings, be then placed in freezing dry
Dry machine is concentrated to dryness, and dried mud is pulverized, and is crossed 80 mesh sieves and is removed root hair, accurately weighs every part of sample 0.5g, turn
Enter into 1.5ml centrifuge tubes, be put into 4 DEG C of Cord bloods, it is to be measured in 24 hours；
3) foreign protein extraction buffer is prepared：100mM Na2CO3, 100mM NaCl, 5mM ethylenediamine tetra-acetic acids, 10mM
Dithiothreitol (DTT), 50mM Na4P2O7·10H2O, 1% (v/v) Triton X-100, pH value is 10, is as extracted slow
Fliud flushing；
4) extracting of foreign protein and measure：Respectively by step 1) it is added to foreign protein extracting buffering with the sample in 2)
It is stripped in liquid, then carries out with Enzyme-linked Immunosorbent Assay reagent (ELISA reagents) box the measure of foreign protein.
According to above scheme, the separation of planktonic organism in the rice field water layer, including the biological precipitation point of water sampling, trip
From specifically including following steps：
1) control time is determined：Control time is selected in tillering stage, jointing stage, boot stage, florescence and the breast of paddy rice respectively
The ripe phase；
2) the precipitation separation of water sampling and planktonic organism：Using 5 point samplings are to all transgenosis cells and non-turn base
Because cell chooses 5 points respectively, each point adopts water in field 10L with band handle measuring cup, loads after bucket, first filters off water with 25 mesh filtering strainer
Rice Litter-fall and other bulk debris, then with the biological net filtrations of 23#, the residue in screen pack is moved into 1L planktonic organisms and precipitated
Device, 1L is settled to water, is precipitated 4 hours；Supernatant liquor is gone into 1L volumetric flasks, siphon by the water sample after precipitation with siphon method
When, note one end of siphon pipe being placed on liquid level little further, coutroi velocity, slowly siphon, to prevent suctioning out settling vessel bottom
Particle sediment, is settled to 1L again, and water sample containing planktonic organism is made, and (water clear after transfer thoroughly, clearly may be used by planktonic organism
See), it is to be measured.
According to above scheme, the measure of plankton species and abundance, specifically includes following steps in the rice field water layer：
1) plankton species of investigation are determined：Investigation species includes：Protozoa, rotifers, cladocera, oar foot
Class, aquatic insect class；
2) counting of protozoa：Planktonic organism water sample will be contained fully to shake up, 0.1ml injection blood count frames are suctioned out
In, covered carries out full sheet counting under an optical microscope, and every part of sample count 5 is averaged, then by concentration
Multiple be converted into quantity in 1L water；
3) counting of rotifers, cladocera and Copepods：Planktonic organism water sample will be contained fully to shake up, with the homemade nothings of 1ml
High graduated glass pipette, extract water sample (small aquatic animal quantitative counting pipe), and sealed with homemade sealer, at 40 times
Full pipe counting is carried out under following stereomicroscope, every part of sample count 5 is averaged, is then converted into by the multiple of concentration
Quantity in 1L water；
4) aquatic insect class：With the naked eye directly count.
According to above scheme, the plantation of the transgenic pest-resistant rice is to the method for the safety evaluatio of planktonic organism：
Applied statistics analysis method comparison transgenic rice paddy and non-turn base rice because of field foreign protein concentration difference, plankton species sum
Difference is measured, the parameters such as Biodiversity of Plankton index, evenness index are counted, and be compared with conventional rice field, final basis
Security of the plantation of statistic analysis result comprehensive analysis transgenic pest-resistant rice to aquatile.
Numerous studies show that transgenosis foreign protein can be from genetically modified crops root system and the species that wither and fall be discharged into environment, water
Need to keep water layer between rice field, and determine concentration and dispersion ability of the transgenic paddy rice foreign protein in water layer and remain in sky
In vain.Each test unit of the present invention is designed to upstream cell and downstream cell, and upstream plantation transgenic paddy rice, downstream plantation is non-
Transgenic paddy rice, downstream non-transgenic paddy rice is divided into two classes, and a class is directly irrigated with source water, another kind of with flowing through transgenosis water
The water of rice cell is irrigated.The foreign protein in transgenic rice paddy water layer and mud can be determined by being so designed that, can also determine outer
Whether source protein can downstream spread with irrigation water.
The universal method for determining foreign protein expression quantity in plant tissue is ELISA method, and this method specificity is good, sensitivity
Height, determines one of sensitivity height key factor extraction buffer.Conventional extraction buffer compound method is：500ml is taken to bore
Shape bottle is separately added into：ddH2O 400ml, NaCl 4g, Na2HPO40.72g, KH2PO40.12g, KCl 0.1g to pH value be 7.4,
Plus ddH2O to 500ml, is then evenly distributed to two 250ml conical flasks, is separately added into 1.125ml Tween20, as extracts
Buffer solution.Such a extraction buffer from plant tissue suitable for extracting foreign protein, to foreign protein in soil and water body
Extraction efficiency is low.Foreign protein extraction buffer [the 100mM Na that the present invention is designed2CO3, 100mM NaCl, 5mM ethylenediamines
Tetraacethyl, 10mM dithiothreitol (DTT)s, 50mM Na4P2O7·10H2O, 1% (v/v) Triton X-100, pH value is
10], extraction efficiency is up to more than 60%.Foreign protein content is atomic in water sample and in mud, does not reach the inspection of ELISA kit
Lower limit is surveyed, the method concentrated to foreign protein that the present invention is designed makes the Monitoring lower-cut of ELISA kit be reduced to originally
1 percent.
Zooplankter individual is small in paddy field, species is more, sensitive to environmental change, is to evaluate the aquatic ring of transgenic pest-resistant rice
The preferable of border security indicates biology.Sampling to water body zooplankter and quantification of more ripe technical side in Hydrobiology
Method, but practice have shown that these methods are not suitable for paddy field zooplankter research, reason has two, and the first paddy field water layer is shallow, the second water
Granule foreign is more in water in field layer, aquatic animal can not be identified and be quantified by general method；In addition, conventional aquatile
The cost for learning, apparatus more complicated to aquatic zooplankter quantitative approach and equipment is high.The present invention is especially set according to paddy field water layer
The method of water intaking body is counted, by tentatively removing zooplankter in the step separation water such as impurity, precipitation and siphon, then with customizing
Pipette carries out Identification of Species and quantitative under stereomicroscope, and method simplicity is with low cost.
The beneficial effects of the invention are as follows：
1) field design method of the invention is applied to the foreign protein of monitoring transgenic pest-resistant rice to Environment release
Concentration, monitoring foreign protein with dispersion of flow scope；
2) present invention is improved foreign protein extraction buffer, coordinates the concentration technique of foreign protein in sample,
The accuracy of detection of ELISA kit can be made to improve 100 times；
3) present invention devises small Planktont counting and quantitative approach in paddy field, it is adaptable to which paddy field water layer is shallow and particle
The characteristics of impurity is more, homemade small aquatic animal quantitative counting pipe enormously simplify the reagent during small aquatic animal quantifies
And apparatus, reduce cost.
Brief description of the drawings
The field that Fig. 1 is the present invention divides schematic diagram.
Embodiment
Technical scheme is illustrated with embodiment below in conjunction with the accompanying drawings.
The present invention provides a kind of method for evaluating transgenic pest-resistant rice aquatic environment security, including following process：
(1) transgenic rice paddy and non-transgenic rice field arrangement and management, comprise the following steps：
1) physical features is smooth, 5 pieces of the convenient field in water source (the repetition evaluation for being used for Environmental security), field area 400- for selection
600m2, aspect ratio range 2: 1 to 1: 1, field periphery and water source are without Sewage Pollution；
2) by step 1) in field be divided into around cell, field build impermeable paddy field ridge and hardening irrigation canals and ditches；It is sudden and violent to prevent
Good gutter is set up on rain flood irrigation, field periphery；The concrete scheme that the field is divided into cell is：Upstream close to water source is small
Area is set up in parallel two pieces of transgenosis cells, and the downstream cell positioned at transgenosis cell downstream is set up in parallel 4 Kuai Fei pavilions gene cells；
Connection irrigation canals and ditches, downstream are set up between the upstream and water source of two pieces of transgenosis cells respectively and non-turns base with middle two pieces respectively
Because cell is connected by irrigation canals and ditches；Two pieces of the outside of the non-transgenic cell is connected by irrigation canals and ditches with the water source respectively；Described 4
Kuai Fei pavilions gene cell sets escape canal respectively (see accompanying drawing 1)；
3) in step 2) in upstream cell plantation transgenic pest-resistant rice, downstream cell plantation non-transgenic paddy rice, in
Between two pieces of non-transgenic cells irrigated with the water in field of upstream transgenosis cell, two pieces of cells of both sides are directly filled with the water in water source
Irrigate；
4) from transplanting to the maturity period, chemical pesticide is not applied, artificial weeding, mid-term not dry field；
(2) in rice field water layer and mud foreign protein measure, including the pretreatment of sample and the extracting of foreign protein with
Determined by ELISA method, specifically include following steps：
1) water sampling and pretreatment：Water sampling cell includes all transgenosis cells and non-transgenic cell, uses
10ml centrifuge tubes carry out field water sampling, and each cell takes 5 pipes, take upper strata clear water, bring back interior, every part of sample is gone to respectively
In new 10ml centrifuge tubes, then freezed, sealed membrane is sealed up after freezing, hole is pricked on film with syringe needle, be then placed in freezing
Drying machine is concentrated to dryness；Dried sample is put into 4 DEG C of Cord bloods, it is to be measured；
2) mud sample collection and pretreatment：Soil sample collection cell includes all transgenosis cells and non-transgenic cell,
Respectively pull out 5 for root and stem of certain plants paddy rice and take rhizosphere mud, load 50ml centrifuge tubes, take back interior and be put into -20 DEG C of refrigerator freezings, be then placed in freezing dry
Dry machine is concentrated to dryness, and dried mud is pulverized, and is crossed 80 mesh sieves and is removed root hair, accurately weighs every part of sample 0.5g, turn
Enter into 1.5ml centrifuge tubes, be put into 4 DEG C of Cord bloods, it is to be measured；
3) foreign protein extraction buffer is prepared：100mM Na2CO3, 100mM NaCl, 5mM ethylenediamine tetra-acetic acids, 10mM
Dithiothreitol (DTT), 50mM Na4P2O7·10H2O, 1% (v/v) Triton X-100, pH value is 10；
4) extracting of foreign protein and measure：Respectively by step 1) it is added to foreign protein extracting buffering with the sample in 2)
It is stripped in liquid, then carries out with ELISA kit the measure of foreign protein；
(3) in the water layer of rice field planktonic organism separation, including the biological precipitation separation of water sampling, trip, specifically include as
Lower step：
1) control time is determined：Control time is selected in tillering stage, jointing stage, boot stage, florescence and the breast of paddy rice respectively
The ripe phase；
2) the precipitation separation of water sampling and planktonic organism：Using 5 point samplings are to all transgenosis cells and non-turn base
Because cell chooses 5 points respectively, each point adopts water in field 10L with band handle measuring cup, loads after bucket, first filters off water with 25 mesh filtering strainer
Rice Litter-fall and other bulk debris, then with the biological net filtrations of 23#, the residue in screen pack is moved into 1L planktonic organisms and precipitated
Device, 1L is settled to water, is precipitated 4 hours；Supernatant liquor is gone into 1L volumetric flasks, siphon by the water sample after precipitation with siphon method
When, note one end of siphon pipe being placed on liquid level little further, coutroi velocity, slowly siphon, to prevent suctioning out settling vessel bottom
Particle sediment, is settled to 1L again, and water sample containing planktonic organism is made, and (water clear after transfer thoroughly, clearly may be used by planktonic organism
See), it is to be measured；
(4) in the water layer of rice field plankton species and abundance measure, specifically include following steps：
1) plankton species of investigation are determined：Investigation species includes：Protozoa, rotifers, cladocera, oar foot
Class, aquatic insect class；
2) counting of protozoa：Planktonic organism water sample will be contained fully to shake up, 0.1ml injection blood count frames are suctioned out
In, covered carries out full sheet counting under an optical microscope, and every part of sample count 5 is averaged, then by concentration
Multiple be converted into quantity in 1L water；
3) counting of rotifers, cladocera and Copepods：Planktonic organism water sample will be contained fully to shake up, with the homemade nothings of 1ml
High graduated glass pipette, extract water sample, and sealed with homemade sealer, managed entirely under stereomicroscope below 40 times
Count, every part of sample count 5 is averaged, is then converted into the quantity in 1L water by the multiple of concentration；
4) aquatic insect with the naked eye directly counts；
(5) safety evaluatio of the plantation of transgenic pest-resistant rice to planktonic organism：Applied statistics analysis method comparison turns
Gene rice field and it is non-turn base rice because of field foreign protein concentration difference, plankton species and quantity variance, statistics planktonic organism is more
The parameters such as sample sex index, evenness index, and be compared with conventional rice field, finally turned according to statistic analysis result comprehensive analysis
Security of the plantation of gene pest-resistant paddy rice to aquatile.
(6) specific evaluation result：
2 bright extensive 63 paddy rice to turn cry1Ab/1Ac and cry2A genes are commented as material under conventional field management pattern
Valency security of the Bt rice to aquatic zooplankter.Wheel animalcule in Bt and non-Bt rice fields water layer, branch angle and oar 3 classes of foot are swum dynamic
Thing has carried out 2 diversity and abundance investigation, and investigation for the first time shows that Bt Tanakas have found 21 zooplankter species, and in non-Bt
Tanaka only identifies 4 kinds, identifies 24 zooplankter species in Bt Tanakas for the second time, rather than Bt Tanakas identify 16 kinds；
Zooplankton abundance occurs in that pole significant difference, the abundance difference of non-Bt Tanakas between Bt fields and non-Bt fields in investigating twice
Fewer than the abundance of Bt Tanakas 95% and 80%.
The transgenosis of table 1 and non-transgenic paddy rice cell Zooplankton Species and abundance investigation result
Above example is only used to illustrative and not limiting technical scheme, although above-described embodiment enters to the present invention
Detailed description is gone, the person skilled of this area should be understood：The present invention can be modified or replaced on an equal basis, but
Not departing from any modification and local replacement of spirit and scope of the invention all should cover in scope of the presently claimed invention.
Claims (6)
1. a kind of method for evaluating transgenic pest-resistant rice aquatic environment security, it is characterised in that including following process：
（1）Transgenic rice paddy and non-transgenic rice field arrangement and management；
（2）The measure of foreign protein in rice field water layer and mud；
（3）The separation of planktonic organism in the water layer of rice field；
（4）The measure of plankton species and abundance in the water layer of rice field；
（5）Safety evaluatio of the plantation of transgenic pest-resistant rice to planktonic organism；
Transgenic rice paddy and non-transgenic rice field arrangement described above and management comprise the following steps：
1）Selection physical features is smooth, 3 to 5 pieces of the convenient field in water source, field area 400-600m2, aspect ratio range 2: 1 to 1: 1,
Field periphery and water source are without Sewage Pollution；
2）By step 1）In field be divided into around cell, field build impermeable paddy field ridge and hardening irrigation canals and ditches；To prevent heavy rain from overflowing
Fill, good gutter is set up on field periphery；
3）In step 2）In upstream cell plantation transgenic pest-resistant rice, downstream cell plantation non-transgenic paddy rice, centre two
Block non-transgenic cell is irrigated with the water in field of upstream transgenosis cell, and two pieces of cells of both sides are directly irrigated with the water in water source；
4）From transplanting to the maturity period, chemical pesticide is not applied, artificial weeding, mid-term not dry field.
2. the method according to claim 1 for evaluating transgenic pest-resistant rice aquatic environment security, it is characterised in that institute
State field and be divided into the concrete scheme of cell and be：Two pieces of transgenosis cells are set up in parallel close to the upstream cell at water source, positioned at turning
The downstream cell in gene cell downstream is set up in parallel 4 pieces of non-transgenic cells；The upstream and water source of two pieces of transgenosis cells
Between set up connection irrigation canals and ditches, downstream respectively and connected respectively with two pieces of middle non-transgenic cells by irrigation canals and ditches；It is described non-to turn base
Because two pieces of the outside of cell is connected by irrigation canals and ditches with the water source respectively；4 pieces of non-transgenic cells set gutter respectively
Canal.
3. the method according to claim 1 for evaluating transgenic pest-resistant rice aquatic environment security, it is characterised in that institute
The measure of foreign protein in rice field water layer and mud, including the pretreatment of sample and the extracting of foreign protein are stated with exempting from by enzyme-linked
Epidemic disease determination of adsorption method, specifically includes following steps：
1）Water sampling and pretreatment：Water sampling cell include all transgenosis cells and non-transgenic cell, with 10ml from
Heart pipe carries out field water sampling, and each cell takes 5 pipes, take upper strata clear water, bring back interior, every part of sample gone to new respectively
In 10ml centrifuge tubes, then freezed, sealed membrane is sealed up after freezing, hole is pricked on film with syringe needle, be then placed in freeze-drying
Machine is concentrated to dryness；Dried sample is put into 4 DEG C of Cord bloods, it is to be measured in 24 hours；
2）Mud sample is gathered and pre-processed：Soil sample collection cell includes all transgenosis cells and non-transgenic cell, respectively pulls out 5
Root and stem of certain plants paddy rice takes rhizosphere mud, loads 50ml centrifuge tubes, takes back interior and be put into -20 DEG C of refrigerator freezings, be then placed in freeze drier
It is concentrated to dryness, dried mud is pulverized, is crossed 80 mesh sieves and remove root hair, accurately weigh every part of sample 0.5g, be transferred to
In 1.5ml centrifuge tubes, 4 DEG C of Cord bloods are put into, it is to be measured in 24 hours；
3）Foreign protein extraction buffer is prepared：250ml conical flasks are taken to be separately added into：100 mM Na2CO3, 100 mM NaCl, 5
MM ethylenediamine tetra-acetic acids, 10 mM dithiothreitol (DTT)s, 50 mM Na4P2O7·10H2O, 1% (v/v) polyethylene glycol octyl phenyl
Ether, pH values are 10, as extraction buffer；
4）The extracting of foreign protein and measure：Respectively by step 1）With 2）In sample be added in foreign protein extraction buffer
It is stripped, then carries out with enzyme-linked immunosorbent assay kit the measure of foreign protein.
4. the method according to claim 1 for evaluating transgenic pest-resistant rice aquatic environment security, it is characterised in that institute
The separation of planktonic organism in the water layer of rice field, including water sampling, the precipitation separation of trip biology are stated, following steps are specifically included：
1）Determine control time：Control time is selected in tillering stage, jointing stage, boot stage, florescence and the milk stage of paddy rice respectively；
2）The precipitation separation of water sampling and planktonic organism：It is small to all transgenosis cells and non-transgenic using 5 point samplings
Area chooses 5 points respectively, and each point adopts water in field 10L with band handle measuring cup, loads after bucket, and first filtering off paddy rice with 25 mesh filtering strainer withers
Junk and other bulk debris, then with the biological net filtrations of 23#, the residue in screen pack is moved into 1L planktonic organism settling vessels,
1L is settled to water, is precipitated 4 hours；Supernatant liquor is gone into 1L volumetric flasks by the water sample after precipitation with siphon method, during siphon,
Note one end of siphon pipe being placed on liquid level little further, coutroi velocity, slowly siphon, to prevent suctioning out the particle of settling vessel bottom
Sediment, is settled to 1L again, and water sample containing planktonic organism is made, to be measured.
5. the method according to claim 1 for evaluating transgenic pest-resistant rice aquatic environment security, it is characterised in that institute
The measure of plankton species and abundance in the water layer of rice field is stated, following steps are specifically included：
1）It is determined that the plankton species of investigation：Investigation species includes：Protozoa, rotifers, cladocera, Copepods, water
Raw insects；
2）The counting of protozoa：Planktonic organism water sample will be contained fully to shake up, suctioned out in 0.1ml injection blood count frames,
Covered, carries out full sheet counting under an optical microscope, and every part of sample count 5 is averaged, then by times of concentration
Number is converted into the quantity in 1L water；
3）The counting of rotifers, cladocera and Copepods：Planktonic organism water sample will be contained fully to shake up, it is homemade without height quarter with 1ml
Spend glass pipet and draw water sample, and sealed with homemade sealer, carry out full pipe counting under stereomicroscope below 40 times,
Every part of sample count 5, averages, is then converted into the quantity in 1L water by the multiple of concentration；
4）Aquatic insect class：With the naked eye directly count.
6. the method according to claim 1 for evaluating transgenic pest-resistant rice aquatic environment security, it is characterised in that institute
The plantation for stating transgenic pest-resistant rice is to the method for the safety evaluatio of planktonic organism：Applied statistics analysis method comparison turns base
Because rice field and it is non-turn base rice because of field foreign protein concentration difference, plankton species and quantity variance, statistics planktonic organism is various
Sex index, evenness index parameter, and be compared with conventional rice field, finally according to statistic analysis result comprehensive analysis transgenosis
Security of the plantation of insect-proof rice to aquatile.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510470544.9A CN104982241B (en) | 2015-08-04 | 2015-08-04 | A kind of method for evaluating transgenic pest-resistant rice aquatic environment security |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510470544.9A CN104982241B (en) | 2015-08-04 | 2015-08-04 | A kind of method for evaluating transgenic pest-resistant rice aquatic environment security |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104982241A CN104982241A (en) | 2015-10-21 |
| CN104982241B true CN104982241B (en) | 2017-10-24 |
Family
ID=54294260
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510470544.9A Expired - Fee Related CN104982241B (en) | 2015-08-04 | 2015-08-04 | A kind of method for evaluating transgenic pest-resistant rice aquatic environment security |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104982241B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114431179A (en) * | 2022-02-25 | 2022-05-06 | 生态环境部南京环境科学研究所 | Evaluation method for safety influence of Bt insecticidal protein on daphnia magna |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102948399A (en) * | 2012-04-13 | 2013-03-06 | 环境保护部南京环境科学研究所 | Daphnia magna breeding method for evaluating safety of transgenic rice |
-
2015
- 2015-08-04 CN CN201510470544.9A patent/CN104982241B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102948399A (en) * | 2012-04-13 | 2013-03-06 | 环境保护部南京环境科学研究所 | Daphnia magna breeding method for evaluating safety of transgenic rice |
Non-Patent Citations (4)
| Title |
|---|
| 《转Bt基因水稻安全性评价研究进展》;贾乾涛等;《湖北农业科学》;20050228;第103-107页 * |
| 《转crylAb/c基因水稻外源蛋白环境残留及其对水生生物的影响研究》;张莉;《中国优秀硕士学位论文全文数据库（电子期刊）》;20140815;第25-70页 * |
| 《转基因抗虫水稻生态安全性评价》;赖凤香;《中国优秀硕士学位论文全文数据库（电子期刊）》;20111215;第6-57页 * |
| 《转基因抗虫水稻的安全性及其防范策略》;刘立军等;《生物技术通报》;20090626;第36-39页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104982241A (en) | 2015-10-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Smith et al. | Establishment of Striacosta albicosta (Lepidoptera: Noctuidae) as a primary pest of corn in the Great Lakes region | |
| CN107262388A (en) | Method for rapidly screening low-accumulation heavy metal crop varieties and device used by same | |
| CN104738034B (en) | Acetic acid cis-3-hexenyl ester synergistic holotrichia parallela sex attractant | |
| CN101849534A (en) | Separation and detection device of entomopathogenic nematode, separation method of entomopathogenic nematode in soil and measuring method of population density | |
| CN111758671A (en) | Method for preventing and treating spodoptera frugiperda by using trichogramma weberiana | |
| Poinar Jr et al. | Survival and horizontal movement of infective stage Neoaplectana carpocapsae in the field | |
| Harbach et al. | A mechanistic approach to assessing the potential for cover crops to serve as trap crops for the soybean cyst nematode | |
| Becker | Quantification of onion vesicular-arbuscular mycorrhizae and their resistance to Pyrenochaeta terrestris. | |
| CN104982241B (en) | A kind of method for evaluating transgenic pest-resistant rice aquatic environment security | |
| CN106124249B (en) | A kind of paddy field wetland water sample acquisition device and acquisition water sample processing method | |
| CN103435163B (en) | Method for de-eutrophicating sewage draining exits of residential communities | |
| Horowitz | Competitive effects of Cynodon dactylon, Sorghum halepense and Cyperus rotundus on cotton and mustard | |
| Ossowski | THE BIOLOGICAL CONTROL OF THE WATTLE BAGWORM (KOTOCHALIA JUNODI HEYL.) BY A VIRUS DISEASE: SMALL‐SCALE PILOT EXPERIMENTS | |
| CN204518596U (en) | A kind of pesticide water ecological risk assessment experimental provision | |
| Speijer et al. | Rate of nematode infestation of clean banana planting material (Musa spp. AAA) in Uganda | |
| Verdejo-Lucas et al. | The citrus nematode: Tylenchulus semipenetrans. | |
| WhITEMAN et al. | Lentic beetles of the Missouri prairie region: habitat and regional associations, with keys to the Hydradephaga | |
| CN104584892A (en) | Experiment device and experiment method for evaluating risk of pesticide to water ecology | |
| Mihajlović et al. | Sclerotinia minor as a new pathogen of lettuce in Serbia | |
| Falloon | Baiting, pathogenicity, and distribution of Phytophthora megasperma var. sojae in New Zealand asparagus soils | |
| Kozhevnikova | Semi-winged pests of cultural plants in Fergana, Uzbekistan | |
| Stirling | MOGGILL QLD 4070 | |
| Nicol | Advisory Services for Nematode Pests | |
| Aish et al. | A potential biocontrol and PGPR activities of bacteria Providencia vermicola against | |
| Tatalovic | Influence of Heterodera glycines infection, plant age and water availability on foliar symptoms of sudden death syndrome, root symptom severity and root morphology of soybean plants infected by Fusarium virguliforme |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20171024 |