CN106290716A - Detect contaminated Folium Mori toxicity and the method for safety - Google Patents
Detect contaminated Folium Mori toxicity and the method for safety Download PDFInfo
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Abstract
The invention discloses and a kind of detect contaminated Folium Mori toxicity and the method for safety, bred silkworms by Folium Mori and test, utilizing test Folium Mori to raise healthy silkworm larva, test general toxicity, neurotoxicity, organ toxicity, development toxicity, genotoxicity and pathogenic microorganism are pathogenic.Compared with general detection method, the present invention is the easiest quickly, reliable results, the most comprehensive, sensitive and accurate, has higher practical value.The toxicity of application present invention acquisition and safety evaluatio result, it is available for Folium Mori as poultry and livestock feed safety evaluatio reference, with confession Folium Mori as edible, the safety evaluatio reference of pharmaceutical products, for contaminated Folium Mori as feedstuff to silkworm health hazard and cultivation risk assessment.Therefore, the present invention utilizes Folium Mori sericulture biologic test, measures toxicity and evaluates safety, and technology path is feasible, has a extensive future.
Description
Technical field
The invention belongs to drug and food safety and silkworm cultivation field, particularly relate to a kind of detect contaminated Folium Mori toxicity and
The method of safety.
Background technology
Mulberry is perennial woody plant, and mulberry leaf nutrient enriches, containing about crude protein 24%-28%, total sugar in dry
23%-26%, crude fat 3.5%-6.0%, necessary rich in dietary fiber, aminoacid, vitamin and many animals and human body
Trace element, containing alkaloid, flavone isoreactivity composition.Folium Mori are unique plant feeds of silkworm, are the bases of Bombyx bombycis production；Mulberry
Leaf also can be as poultry animal feed.Folium Mori are classified as and are food and medicine by Ministry of Public Health.Folium Mori are Chinese crude drugs, containing 1-deoxidation
Alkaloid, the flavone isoreactivity compositions such as nojirimycin (DNJ), have the drug effects such as blood sugar lowering, blood fat reducing and blood pressure lowering.Folium Mori can make
Folium Mori dish, has delicate fragrance, the feature that fresh and crisp, color and luster is dark green, is to have better nutritivity to be worth and the green vegetables of health care；Mulberry
Leaf can make Folium Mori tea, and millet paste is limpid bright, and delicate fragrance is sweet, and fresh alcohol is tasty and refreshing, to blood sugar lowering, the skin ulcer that disappears speckle dispelling, fat-reducing fat eliminating, reduction
Blood is stained with certain effect；Powder of Folium Mori also can make Folium Mori noodles, Folium Mori cake etc., as the food of the special populations such as diabetes.
Mulberry field and neighbouring crops use chemical pesticide, Folium Mori can be made contaminated and contain residual toxicity；Muscardine pesticide,
The biological pesticides such as the insect pathogenic bacterias such as Bacillus thuringiensis pesticide, nuclear polyhedrosis virus (fungus, antibacterial, virus) preparation, Sang Fen
The Mulberry Pests such as louse, diaphania can occur the disease that silkworm is same, and ill insect bodies, Excreta, palea etc. pollute Folium Mori, all can become
The source of infection broken out for flacherie；Folium Mori are caused harm by pathogenic fungi, make Folium Mori by fungal contamination, some funguses and secondary metabolite thereof
Containing mycotoxin；Plant gas atmosphere pollution, sulfide, fluoride etc., by Folium Mori absorption accumulation, make Folium Mori dirty by poisonous substance
Dye, utilizes to Folium Mori and brings unsafe factor.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of simplicity quickly, reliable results, the most comprehensive, sensitive and accurate
Detect contaminated Folium Mori toxicity and the method for safety, it is adaptable to contaminated Folium Mori as feedstuff to silkworm health hazard and cultivation
Risk assessment.
For solve above-mentioned technical problem, the present invention by the following technical solutions:
Detect contaminated Folium Mori toxicity and the method for safety, bred silkworms by Folium Mori and test, utilize test Folium Mori to raise strong
Health silkworm larva, according to action, figure, food Mulberry, the defecation of silkworm, weave silk, pupate, change moth, copulation, lay eggs, skin, growth promoter
Aspect difference, and mortality rate, larva pupa rate, larva become moth rate, normal spawning moth rate, good ovum rate index, the general poison of test
Property, neurotoxicity, organ toxicity, development toxicity, genotoxicity and pathogenic microorganism are pathogenic.
Above-mentioned detection contaminated Folium Mori toxicity and the method for safety, comprise the following steps:
(1) healthy test silkworm is prepared；(2) pluck test Folium Mori subregion to breed silkworms；(3) health status of silkworm is observed in investigation；
(4) general toxicity test；(5) neurotoxicity test；(6) organ toxicity's test；(7) development toxicity test；
(8) genotoxicity test；(9) the pathogenic test of pathogenic microorganism；(10) test Folium Mori are as the peace of feed of mulberry silkworm
Full property overall merit.
Step (1) is carried out by following operation: select qualified silkworm egg, and breed silkworms routinely operation and sterilizing prevent disease measure, to institute
With test silkworm room and apparatus carries out disinfection, hatching of silkworm egg, young silkworm raise；1 age～2 age young silkworm raise with normal Folium Mori, raise temperature
Spend 27 DEG C-28 DEG C, relative humidity 85%-90%, long-day；With playing silkworm in 2 ages～arbitrary age in 4 ages as tested silkworm, process is set
District, raises district, normal Folium Mori raising district, every treatment region three replicated plot including test Folium Mori, and every community is same head number for examination silkworm
More than 20.
Step (2) is carried out by following operation: pluck the test Folium Mori and normal Folium Mori specified in corresponding treatment region
The raising of test silkworm；By same environmental condition and operation of breeding silkworms, temperature 25 DEG C-27 DEG C, relative humidity 75%-85%, natural light
According to, raise and cocoon to being placed on small straw bundles to spin cocoons.
Step (3) is carried out by following operation: observes and compares test Folium Mori raising district with normal Folium Mori raising district silkworm at body
State, take action, eat Mulberry, defecation, cast off a skin, weave silk, pupate, change moth, copulation, lay eggs, difference in terms of skin, anus, investigation statistics
Mortality rate, strong pupa rate, Cocooning rate, larva pupa rate, larva become moth rate, normal spawning moth rate, good ovum rate index.
Step (4) is carried out to (7) by following operation:
(4) general toxicity test
Occur telling liquid, refusing to eat, body contracting, yaw, body twist, slow-witted volt is motionless, feed the reactionless or dead symptom of leaf, and extremely
Rate of dying test Folium Mori are raised district and are significantly higher than check plot, or strong silkworm rate, larva pupa rate test Folium Mori are raised district and are substantially less than
Check plot, diagnosing tested Folium Mori has general toxicity；
(5) neurotoxicity test
Occur mad dry, disorderly climb, tell the dysautonomia symptom such as sleave, rotation pupa behavior, and mortality rate test Folium Mori are raised district and are shown
Write higher than check plot, or strong silkworm rate, larva pupa rate, larva become moth rate test Folium Mori to raise district to be substantially less than check plot,
Diagnose tested Folium Mori and have neurotoxicity；
(6) organ toxicity's test
There is greyish black mottle or burns shape speckle, non exuviated larva, partly peels silkworm, half decortication pupal cell wall damage in body wall；Middle intestinal or
Anal lesions and occur that proctoptosis, row's loose stool, beads excrement etc. are abnormal, for the performance of organ injury；Appearance organ injury symptom, and extremely
Rate of dying test Folium Mori are raised district and are significantly higher than check plot, or strong silkworm rate, larva pupa rate, larva become moth rate test Folium Mori to raise
District is substantially less than check plot, and diagnosing tested Folium Mori has organ toxicity
(7) development toxicity test
Occur non molting larva, non exuviated larva and do not cocoon, do not pupate, do not sprout wings, the abnormal development such as do not lay eggs, and mortality rate surveys
Examination Folium Mori are raised district and are significantly higher than check plot, or strong silkworm rate, larva pupa rate, that larva becomes moth rate test Folium Mori to raise district is notable
Less than check plot, diagnosing tested Folium Mori has development toxicity.
Step (8) genotoxicity is tested, and carries out by following operation:
(8.1) after cocooing, GPF (General Protection False is sprouted wings to Pupa bombycis, changes moth morning on the same day with the female moth in experimental plot and male moth copulation, hands over
5h-6h between timing, the female moth of separation of copulating moth after vibration urinates, and puts into unilateral tonsillitis in paper bag of laying eggs and lays eggs the production of hybrid seeds, and morning next day, 8:00 killed moth eventually
Only laying eggs, silkworm egg GPF (General Protection False becomes fixing color to silkworm seed in 7 days；
(8.2) investigation do not lay eggs moth, lay eggs moth, the quantity of the abnormal improper moth that lays eggs of moth of fertilization less, calculate improper product
Ovum moth rate；The unilateral tonsillitis laying of investigation normal spawning moth and good ovum number, calculate good ovum rate；
(8.3) improper moth rate of laying eggs, test Folium Mori are raised district and are significantly higher than check plot；Or good ovum rate, test Folium Mori
Raise district be substantially less than check plot, diagnosing tested Folium Mori has genotoxicity.
The pathogenic test of step (9) pathogenic microorganism, is carried out by following operation:
(9.1) test Folium Mori raise district and check plot locellus isolated rearing, are raised by different poultry raiser's picking leaves respectively, prevent
Cross infection flacherie；
(9.2) every 4h～6h observes once, finds that ill silkworm, dead silkworm pick up other raising frame isolation in time, prevents two
Subinfection；
(9.3) sick silkworm, dead silkworm are carried out the diagnosis of infectiousness flacherie kind, a number of dying of illness for all kinds of flacherie of statistic record,
Add up the sickness rate of all kinds of flacherie；
(9.4) certain flacherie symptom occur, and sickness rate test Folium Mori are raised district and are significantly higher than check plot, diagnosis is tested
Folium Mori have pathogenic microorganism pathogenic.
Step (10) test Folium Mori, as the safety overall merit of feed of mulberry silkworm, are carried out by following operation:
(10.1) general toxicity, neurotoxicity, organ toxicity, development toxicity, pathogenic microorganism are pathogenic, as not having after tested
Have 1 toxic, then test Folium Mori for breed silkworms product cocoon be safe；If any 1 or multinomial toxic, then test Folium Mori are used for
It is unsafe for breeding silkworms and producing cocoon；
(10.2) general toxicity, neurotoxicity, organ toxicity, development toxicity, pathogenic microorganism be pathogenic after tested, reproduction
Toxicity, as do not have 1 toxic, then test Folium Mori for parent silkworm rearing breed silkworms and the production of hybrid seeds be safe；If any 1 or multinomial poisonous
Property, then test Folium Mori are bred silkworms for parent silkworm rearing and the production of hybrid seeds is unsafe.
For the safety issue utilizing contaminated Folium Mori to exist, inventor establishes the contaminated Folium Mori toxicity of a kind of detection
With the method for safety, bred silkworms by Folium Mori and test, utilize test Folium Mori to raise healthy silkworm larva, according to action, the body of silkworm
State, food Mulberry, defecation, weave silk, pupate, changes moth, copulation, lay eggs, skin, growth promoter aspect difference, and mortality rate, larva one-tenth
Pupa rate, larva become moth rate, normal spawning moth rate, good ovum rate index, test general toxicity, neurotoxicity, organ toxicity, growth poison
Property, genotoxicity and pathogenic microorganism are pathogenic.Compared with general detection method, the present invention is the easiest quickly, result can
Lean on, the most comprehensive, sensitive and accurate, there is higher practical value.The toxicity of application present invention acquisition and safety evaluatio result,
It is available for Folium Mori as poultry and livestock feed safety evaluatio reference, and for Folium Mori as edible, the safety evaluatio reference of pharmaceutical products, uses
In contaminated Folium Mori as feedstuff to silkworm health hazard and cultivation risk assessment.Therefore, the present invention utilizes Folium Mori sericulture biology
Test, measures toxicity and evaluates safety, and technology path is feasible, has a extensive future.
Detailed description of the invention
One, the present invention detects the basic step of method and the operational approach of contaminated Folium Mori toxicity and safety
(1) healthy test silkworm is prepared
Selecting the qualified silkworm egg of local popularization and application, breed silkworms routinely operation and sterilizing prevent disease measure, to test silkworm used
Room and apparatus carry out disinfection, hatching of silkworm egg, young silkworm raise；1 age～2 age young silkworm raise with normal Folium Mori, rearing temperature 27 DEG C-28
DEG C, relative humidity 85%-90%, long-day (light application time 16h every day)；With playing silkworm in 2 ages～arbitrary age in 4 ages as tested silkworm,
Arranging treatment region, including testing, Folium Mori raise district, normal Folium Mori raise district (check plot), every treatment region three replicated plot, the least
District is equal several more than 20 for examination silkworm.
(2) pluck test Folium Mori subregion to breed silkworms
Pluck the test Folium Mori specified and normal Folium Mori (comparison) in corresponding treatment region, test the raising of silkworm；By same
Sample environmental condition and operation of breeding silkworms, temperature 25 DEG C-27 DEG C, relative humidity 75%-85%, natural lighting, raise and cocoon to being placed on small straw bundles to spin cocoons.
(3) health status of silkworm is observed in investigation
Observation compare test Folium Mori raise district and normal Folium Mori raise district silkworm figure, take action, eat Mulberry, defecation, cast off a skin,
Weave silk, pupate, change moth, copulation, lay eggs, the difference of the aspect such as skin, anus, investigation statistics mortality rate, strong pupa rate, Cocooning rate,
Larva pupa rate, larva become the indexs such as moth rate, normal spawning moth rate, good ovum rate.
(4) general toxicity test
Occur telling liquid, refusing to eat, body contracting, yaw, body twist, slow-witted volt is motionless, feed the reactionless or dead symptom of leaf, and extremely
Rate of dying test Folium Mori are raised district and are significantly higher than (the p ＜ 0.05) of check plot, or strong silkworm rate, larva pupa rate test Folium Mori raise district
Being substantially less than (the p ＜ 0.05) of check plot, diagnosing tested Folium Mori has general toxicity；
(5) neurotoxicity test
The mad dysautonomia symptom climbing, telling the behaviors such as sleave, rotation pupa dry, random occurs, and mortality rate test Folium Mori raise district
It is significantly higher than (the p ＜ 0.05) of check plot, or strong silkworm rate, that larva pupa rate, larva become moth rate test Folium Mori to raise district is the lowest
In (the p ＜ 0.05) of check plot, diagnosing tested Folium Mori has neurotoxicity；
(6) organ toxicity's test
There is greyish black mottle or burns shape speckle, non exuviated larva, partly peels the body wall damage such as silkworm, half decortication pupa in body wall；Middle intestinal
Or anal lesions and occur that proctoptosis, row's loose stool, beads excrement etc. are abnormal, for the performance of organ injury；Organ injury symptom occurs, and
Mortality rate test Folium Mori are raised district and are significantly higher than (the p ＜ 0.05) of check plot, or strong silkworm rate, larva pupa rate, larva become moth rate
Test Folium Mori are raised district and are substantially less than (the p ＜ 0.05) of check plot, and diagnosing tested Folium Mori has organ toxicity
(7) development toxicity test
Occur non molting larva, non exuviated larva and do not cocoon, do not pupate, do not sprout wings, the abnormal development such as do not lay eggs, and mortality rate surveys
Examination Folium Mori are raised district and are significantly higher than (the p ＜ 0.05) of check plot, or strong silkworm rate, larva pupa rate, larva become moth rate to test Folium Mori
Raising district and be substantially less than (the p ＜ 0.05) of check plot, diagnosing tested Folium Mori has development toxicity.
(8) genotoxicity test
(8.1) after cocooing, GPF (General Protection False is sprouted wings to Pupa bombycis, changes moth morning on the same day with the female moth in experimental plot and male moth copulation, hands over
5h-6h between timing, the female moth of separation of copulating moth after vibration urinates, and puts into unilateral tonsillitis in paper bag of laying eggs and lays eggs the production of hybrid seeds, and morning next day, 8:00 killed moth eventually
Only laying eggs, silkworm egg GPF (General Protection False becomes fixing color to silkworm seed in 7 days；
(8.2) investigation do not lay eggs moth, lay eggs that (laying is less than the Bombycis mori of 120 or less than this kind normal spawning for moth less
The Bombycis mori of several 30%), the quantity of the improper moths that lay eggs such as the abnormal moth of fertilization (produced silkworm seed 80% is above nonfertilization ovum etc.), count
Calculate improper moth rate of the laying eggs ratio of female moth number of always participating in the experiment (the most improper moth number of laying eggs account for)；The unilateral tonsillitis of investigation normal spawning moth produces
Ovum number and good ovum number, calculate good ovum rate；
(8.3) improper moth rate of laying eggs, test Folium Mori are raised district and are significantly higher than (the p ＜ 0.05) of check plot；Or good ovum rate,
Test Folium Mori raising district is substantially less than (the p ＜'s 0.05) of check plot, and diagnosing tested Folium Mori has genotoxicity.
(9) the pathogenic test of pathogenic microorganism
(9.1) test Folium Mori raise district and check plot locellus isolated rearing, are raised by different poultry raiser's picking leaves respectively, prevent
Cross infection flacherie；
(9.2) every 4h～6h observes once, finds that ill silkworm, dead silkworm pick up other raising frame (dish) isolation in time, anti-
Only superinfection；
(9.3) sick silkworm, dead silkworm are carried out the diagnosis of infectiousness flacherie kind, a number of dying of illness for all kinds of flacherie of statistic record,
Add up the sickness rate of all kinds of flacherie；
(9.4) certain flacherie symptom occurs, and sickness rate test Folium Mori is raised district and are significantly higher than (the p ＜ 0.05) of check plot,
Diagnosing tested Folium Mori has pathogenic microorganism pathogenic.
(10) test Folium Mori are as the safety overall merit of feed of mulberry silkworm
(10.1) general toxicity, neurotoxicity, organ toxicity, development toxicity, pathogenic microorganism are pathogenic, as not having after tested
Have 1 toxic, then test Folium Mori for breed silkworms product cocoon be safe；If any 1 or multinomial toxic, then test Folium Mori are used for
It is unsafe for breeding silkworms and producing cocoon；
(10.2) general toxicity, neurotoxicity, organ toxicity, development toxicity, pathogenic microorganism be pathogenic after tested, reproduction
Toxicity, as do not have 1 toxic, then test Folium Mori for parent silkworm rearing breed silkworms and the production of hybrid seeds be safe；If any 1 or multinomial poisonous
Property, then test Folium Mori are bred silkworms for parent silkworm rearing and the production of hybrid seeds is unsafe.
Two, the present invention detects the application example of method of contaminated Folium Mori toxicity and safety
Following example basic step and operational approach are carried out with reference to one.
Application examples 1 band poison Chemical Mixed Fertilizer uses toxicity and the safety evaluatio of the Folium Mori in mulberry field
Certain sericulture village newly pushes away certain brand Chemical Mixed Fertilizer (be called for short A fertilizer), and A fertilizer is qualified products, have " fertilizer registration card ",
The certificate such as " production licence ", " work certificate ", A Fertilizer nutrient is complete, fertilizer efficiency is stable in fertilizer plant publicity, and volume increase is substantially, anti-
Worm power is strong.Mid-May in the first half of the year, this fertilizer was used in this mulberry field, village 50%.3 batches of silkworms that this village's late May to late July raises, portion
The silkworm dividing sericulture family is poisoned, and production incurs loss.It is fertile that sericulture family suspects that A fertilizer is probably medicine, may contain poisonous in fertilizer
Thing (insecticide), is applied to mulberry field and makes Folium Mori contaminated zone poison.But fertilizer plant denies that A fertilizer is containing Toxic.Sampling A fertilizer carries out pesticide
Residue detection, does not detect common pesticides composition.
This mulberry field, village late July in whole summer is cut down, and is unified at 10cm～20cm of ground to cut and cuts down, and carry out mulberry field weeding,
Clear garden and fertilising, stop using A fertilizer.
Materials and methods: the most poisonous for the test Folium Mori second half year, the Folium Mori in this mulberry field, plot, village 3 are surveyed by mid or late September
Prelibation.3 mulberry field, plot code names are: A1 mulberry field, A2 mulberry field, CK mulberry field.Wherein, A1 mulberry field, A2 mulberry field use A fertile the first half of the year
, there is poisoning of breeding silkworms in material；CK mulberry field does not use A fertilizer the first half of the year, breeds silkworms and intoxicating phenomenon does not occur.Treatment region is set:
A1 mulberry field Folium Mori raise district, A2 mulberry field Folium Mori raise district, CK mulberry field Folium Mori raise district, and every treatment region 3 communities of breeding silkworms (i.e. repeat 3
District), 50, each community silkworm.For examination silkworm it is: the former silkworm 3 of silkworm " 7532 " kind plays silkworm, silkworm room temperature 25 DEG C-27 DEG C, phase age
To temperature 75%-85%, natural lighting.Raise with test Folium Mori continuously and be placed on small straw bundles to spin cocoons cocoon to five matured silkworm phases in age, a point community for 13 days, adjust
Look into and record each age death head number, dead head number in the 7th day investigation cocoon after being placed on small straw bundles to spin cocoons.
Result (table 1): A1 mulberry field Folium Mori are raised district and started to occur successively poisoning symptom from the 2nd day, and A2 mulberry field Folium Mori are raised
There is poisoning symptom from the 5th day in district.Poisoning symptom show: tell liquid, refusing to eat, body contracting, yaw, body twist, death, spasm,
Disorderly climb, after death corpse is C-shaped, distortion, linear, S-shaped various shape.Play silkworm 3 ages to the 7th day cumulative mortality after being placed on small straw bundles to spin cocoons, A1
Mulberry field Folium Mori are raised district and are reached 55.33%, and pole is significantly higher than normal mulberry field Folium Mori and raises district；A2 mulberry field Folium Mori are raised district and are reached
18.67%, pole is significantly higher than normal mulberry field Folium Mori and raises district.
Conclusion: to mid or late September, A1 mulberry field, the Folium Mori in A2 mulberry field have general toxicity and neurotoxicity, are not for sericulture
Safety.
The death condition that table 1 is bred silkworms with test Folium Mori
Application examples 2 mulberry whitefly severe causes harm the toxicity of Folium Mori and safety evaluatio.
In certain year June, a mulberry field is had seriously to be caused harm by mulberry whitefly, the close and numerous nymph (children being covered with mulberry whitefly of blade back
Worm).For measure mulberry whitefly harm Folium Mori toxicity with pathogenic, test as follows:
Materials and methods: pluck the 9th leaf position blade that mulberry whitefly causes harm heavier, the grey white point of blade back is more (severe causes harm leaf,
Every cm2Have more than 8 mulberry whitefly nymphs or corpse) test；For examination silkworm: Silkworm varieties " 7532 × 932 ", conventional raising caused for 3 ages
Play silkworm.Treatment region is arranged: mulberry whitefly severe causes harm leaf district and normal Folium Mori district (check plot), respectively raises in a silkworm room, prevents
Mutually infect.Each treatment region is bred silkworms 3 communities (repeating 3 communities), 30, every community silkworm.Feed mulberry whitefly after playing silkworm 3 ages to cause harm Folium Mori
To 4 latter stages in age (totally 8 days).
Result: mulberry whitefly severe leaf district of causing harm feeds after leaf silkworm body after 36h and i.e. has scab to occur, almost 100% silkworm after 48h
Diseased body speckle, wherein the foot of the silkworm of 80% has black patches.Having 90% silkworm not fall asleep and dead when age, minority is fallen asleep energy
Cast off a skin, continue to feed aleyrodid Ye Youhui of causing harm and again grow scab, dead in 4 age ends.Cumulative mortality 100%.Normal Folium Mori
District (check plot) mortality rate is 0.Mulberry whitefly is caused harm the silkworm corpse in leaf district, picks up at the culture dish being lined with wet filter paper, continues to observe:
After death 5h, the cadaveric rigidity of 100%, after 2 days, corpse surface is covered with white hypha, and after death 4d-5d corpse surface is covered with Lycoperdon polymorphum Vitt
Powder.After diagnosing, dead silkworm is that silkworm ash deadlock is sick and lethal.
Conclusion: mulberry whitefly leaf of causing harm has an organ toxicity, there will be black scab, affect hibernating worm and cast off a skin after the contact of silkworm body；
There is the pathogenic of silkworm ash deadlock disease；With mulberry whitefly cause harm leaf breed silkworms be unsafe.
Application examples 3 mulberry brown leaf rust causes harm the toxicity of Folium Mori and safety evaluatio
Mulberry brown leaf rust (Aecidium mori Barclay), also known as red powder disease, Jin Sang, gold leaf, is common true of mulberry
Fungus diseases, mainly the cause harm tender shoots of mulberry young sprout, blade and Flos Mori, mulberry.After Folium Mori are caught an illness, it may appear that the gyalectiform rust of spot distribution
Spore device, the aecidiospore spraying orange yellow powder from aecidium is covered with Folium Mori.
For evaluating toxicity and the safety of the Folium Mori polluted by brown leaf rust fungus and secondary metabolite thereof, carry out test as follows:
Materials and methods: for examination silkworm: Silkworm varieties is Guangdong and Guangxi Provinces two, raises routinely and plays silkworm to 3 ages.If treatment region: red rust
Seriously ill degree is caused harm, and leaf raises district, normal leaf raises district (check plot), and each treatment region raises 3 communities (3 replicated plot), every community 50
Head silkworm.From 3 ages, silkworm starts, and raises to cocooing with test leaf continuously.By " mulberry silk cocoon brings up silkworm technical regulation " (DB45/
T87-2003) environmental condition and requirement carry out breeding silkworms and Bombyx bombycis protection, and pupa time, moth phase, the protection temperature of ovum phase are 24 DEG C-26
DEG C, relative humidity 75%-85%.
Survey items: (1) nibble Mulberry, creep, active situation of cocooing of weaving silk；(2) the organ feelings such as silkworm body wall, middle intestinal, mouthpart
Condition；(3) silkworm growth promoter, hibernating worm, matured silkworm, cocoon, pupate, change moth situation；(4) investigation non molting larva, non exuviated larva, knot of not weaving silk
Cocoon silkworm, do not pupate the abnormal development silkworms such as silkworm；(5) investigation do not lay eggs moth, lay eggs the moth (Bombycis mori that laying ＜ is 120, or lay eggs less
The Bombycis mori of number these kind normal spawning numbers 30% of ＜), the anon-normal such as the abnormal moth of fertilization (produced silkworm seed 20% is above nonfertilization ovum)
Often lay eggs the quantity of moth, calculate improper moth rate of the laying eggs ratio of female moth number of always participating in the experiment (the most improper moth number of laying eggs account for)；Investigation
The unilateral tonsillitis laying of normal spawning moth and good ovum number, calculate good ovum rate；(6) a situation arises for investigation infectiousness flacherie
Result (table 2) and evaluation:
(1) brown leaf rust severe cause harm leaf district do not occur telling the silkworms such as liquid, refusing to eat, body contracting, yaw, body twist acute in
Toxication shape, strong silkworm rate is 100%, Cocooning rate 100%, dead cage rate be 0,3 age silkworm pupa rate 100%, 3 silkworm in age become moth rate high
97.33%, and higher than check plot (normal leaf), diagnosis: brown leaf rust severe leaf district of causing harm does not has general toxicity；
(2) to cause harm that leaf district silkworm does not occur that slow-witted volt is motionless, to feed leaf reactionless or mad dry, disorderly climb, disorderly tell for brown leaf rust severe
Silk, the rotation dysautonomia silkworm such as pupa, diagnosis: brown leaf rust severe leaf district of causing harm does not has neurotoxicity；
(3) brown leaf rust severe leaf district silkworm body wall of causing harm does not has that abnormal scab, defecation are normal, it is normal to weave silk, molting is just being peeled
Often, pupate normal, show that brown leaf rust severe leaf district of causing harm does not has organ toxicity；
(4) brown leaf rust severe cause harm leaf district do not occur non molting larva, non exuviated larva, do not weave silk cocoon silkworm, do not pupate silkworm etc.
Abnormal development silkworm, 3 age silkworm pupa rate 100%, 3 age silkworm become moth rate to be up to 97.33%, and higher than check plot, diagnosis: brown leaf rust weight
Spend leaf of causing harm and there is no development toxicity；
(5) the brown leaf rust severe improper moth rate of laying eggs in leaf district that causes harm is only 6.67%, and less than check plot；Unilateral tonsillitis good ovum rate
Up to 99.66%, higher than check plot, diagnosis: brown leaf rust severe leaf of causing harm does not has genotoxicity；
(6) brown leaf rust severe cause harm leaf district be good for silkworm rate, 3 age silkworm pupa rate 100%, the silkworm that dies of illness there is not, diagnosis: red rust
Seriously ill degree leaf of causing harm does not has pathogenic microorganism pathogenic.
Conclusion: brown leaf rust severe leaf of causing harm is bred silkworms and do not had the pathogenic of toxicity and pathogenic microorganism, is safe.
Table 2 brown leaf rust severe cause harm leaf breed silkworms vitality and reproductivity
Claims (9)
1. one kind is detected contaminated Folium Mori toxicity and the method for safety, it is characterised in that is bred silkworms by Folium Mori and tests, utilizes and survey
Examination Folium Mori raise healthy silkworm larva, according to action, figure, food Mulberry, the defecation of silkworm, weave silk, pupate, change moth, copulation, lay eggs,
Skin, growth promoter aspect difference, and mortality rate, larva pupa rate, larva become moth rate, normal spawning moth rate, good ovum rate to refer to
Mark, test general toxicity, neurotoxicity, organ toxicity, development toxicity, genotoxicity and pathogenic microorganism are pathogenic.
Detect contaminated Folium Mori toxicity and the method for safety the most according to claim 1, it is characterised in that include following step
Rapid:
(1) healthy test silkworm is prepared；(2) pluck test Folium Mori subregion to breed silkworms；(3) health status of silkworm is observed in investigation；
(4) general toxicity test；(5) neurotoxicity test；(6) organ toxicity's test；(7) development toxicity test；
(8) genotoxicity test；(9) the pathogenic test of pathogenic microorganism；(10) test Folium Mori are as the safety of feed of mulberry silkworm
Overall merit.
Detect contaminated Folium Mori toxicity and the method for safety the most according to claim 2, it is characterised in that step (1) by with
Lower operation is carried out: select qualified silkworm egg, and test silkworm room used and apparatus are carried out by breed silkworms routinely operation and sterilizing prevent disease measure
Sterilization, hatching of silkworm egg, young silkworm are raised；1 age～2 age young silkworm raise with normal Folium Mori, rearing temperature 27 DEG C-28 DEG C, relative humidity
85%-90%, long-day；With playing silkworm in 2 ages～arbitrary age in 4 ages as tested silkworm, treatment region is set, raises including test Folium Mori
District, normal Folium Mori raise district, every treatment region 3 replicated plot, and every community is same several more than 20 for examination silkworm.
Detect contaminated Folium Mori toxicity and the method for safety the most according to claim 2, it is characterised in that step (2) by with
Lower operation is carried out: pluck the test Folium Mori specified and normal Folium Mori test the raising of silkworm in corresponding treatment region；By same
Environmental condition and operation of breeding silkworms, temperature 25 DEG C-27 DEG C, relative humidity 75%-85%, natural lighting, raise and cocoon to being placed on small straw bundles to spin cocoons.
Detect contaminated Folium Mori toxicity and the method for safety the most according to claim 2, it is characterised in that step (3) by with
Lower operation is carried out: observe compare test Folium Mori raise district and normal Folium Mori raisings district silkworm figure, take action, eat Mulberry, defecation, slough off
Skin, weave silk, pupate, change moth, copulation, lay eggs, difference in terms of skin, anus, investigation statistics mortality rate, strong pupa rate, Cocooning rate,
Larva pupa rate, larva become moth rate, normal spawning moth rate, good ovum rate index.
Detect contaminated Folium Mori toxicity and the method for safety the most according to claim 1, it is characterised in that step (4) is extremely
(7) carry out by following operation:
(4) general toxicity test
Occur telling liquid, refusing to eat, body contracting, yaw, body twist, slow-witted volt is motionless, feed the reactionless or dead symptom of leaf, and mortality rate
Test Folium Mori are raised district and are significantly higher than check plot, or strong silkworm rate, larva pupa rate test Folium Mori are raised district and are substantially less than comparison
District, diagnosing tested Folium Mori has general toxicity；
(5) neurotoxicity test
Occur mad dry, disorderly climb, tell sleave, rotation pupa behavior dysautonomia symptom, and mortality rate test Folium Mori to raise district significantly high
In check plot, or strong silkworm rate, larva pupa rate, larva become moth rate test Folium Mori to raise district to be substantially less than check plot, diagnosis
Tested Folium Mori have neurotoxicity；
(6) organ toxicity's test
There is greyish black mottle or burns shape speckle, non exuviated larva, partly peels silkworm, half decortication pupal cell wall damage in body wall；Middle intestinal or anus
Damage and occur that proctoptosis, row's loose stool, beads excrement are abnormal, for the performance of organ injury；Organ injury symptom occurs, and mortality rate is surveyed
Examination Folium Mori are raised district and are significantly higher than check plot, or strong silkworm rate, larva pupa rate, that larva becomes moth rate test Folium Mori to raise district is notable
Less than check plot, diagnosing tested Folium Mori has organ toxicity
(7) development toxicity test
Occur non molting larva, non exuviated larva and do not cocoon, do not pupate, do not sprout wings, abnormal development of not laying eggs, and mortality rate test Folium Mori
Raise district and be significantly higher than check plot, or strong silkworm rate, larva pupa rate, that larva becomes moth rate test Folium Mori to raise district is the most right
According to district, diagnosing tested Folium Mori has development toxicity.
Detect contaminated Folium Mori toxicity and the method for safety the most according to claim 2, it is characterised in that step (8) reproduction
Toxotest, is carried out by following operation:
(8.1) after cocooing, GPF (General Protection False is sprouted wings to Pupa bombycis, changes moth morning on the same day with the female moth in experimental plot and male moth copulation, during copulation
Between 5h-6h, the female moth of separation of copulating moth after vibration urinates, and puts into unilateral tonsillitis in paper bag of laying eggs and lays eggs the production of hybrid seeds, and 8:00 killed moth and terminated producing morning next day
Ovum, silkworm egg GPF (General Protection False becomes fixing color to silkworm seed in 7 days；
(8.2) investigation do not lay eggs moth, lay eggs moth, the quantity of the abnormal improper moth that lays eggs of moth of fertilization less, calculate the improper moth that lays eggs
Rate；The unilateral tonsillitis laying of investigation normal spawning moth and good ovum number, calculate good ovum rate；
(8.3) improper moth rate of laying eggs, test Folium Mori are raised district and are significantly higher than check plot；Or good ovum rate, test Folium Mori are raised
District be substantially less than check plot, diagnosing tested Folium Mori has genotoxicity.
Detect contaminated Folium Mori toxicity and the method for safety the most according to claim 1, it is characterised in that step (9) cause of disease
The pathogenic test of microorganism, is carried out by following operation:
(9.1) test Folium Mori raise district and check plot locellus isolated rearing, are raised by different poultry raiser's picking leaves respectively, prevent from intersecting
Infect flacherie；
(9.2) every 4h～6h observes once, finds that ill silkworm, dead silkworm pick up other raising frame isolation in time, prevents secondary sense
Dye；
(9.3) sick silkworm, dead silkworm are carried out the diagnosis of infectiousness flacherie kind, a number of dying of illness for all kinds of flacherie of statistic record, statistics
The sickness rate of all kinds of flacheries；
(9.4) certain flacherie symptom occurs, and sickness rate test Folium Mori are raised district and are significantly higher than check plot, diagnose tested Folium Mori
There is pathogenic microorganism pathogenic.
Detect contaminated Folium Mori toxicity and the method for safety the most according to claim 2, it is characterised in that step (10) is surveyed
Examination Folium Mori, as the safety overall merit of feed of mulberry silkworm, are carried out by following operation:
(10.1) general toxicity, neurotoxicity, organ toxicity, development toxicity, pathogenic microorganism are pathogenic, as not having 1 after tested
Item is toxic, then test Folium Mori are safe for product cocoon of breeding silkworms；If any 1 or multinomial toxic, then test Folium Mori are used for supporting
It is unsafe that silkworm produces cocoon；
(10.2) after tested general toxicity, neurotoxicity, organ toxicity, development toxicity, pathogenic microorganism is pathogenic, reproduction is malicious
Property, as do not have 1 toxic, then test Folium Mori for parent silkworm rearing breed silkworms and the production of hybrid seeds be safe；If any 1 or multinomial poisonous
Property, then test Folium Mori are bred silkworms for parent silkworm rearing and the production of hybrid seeds is unsafe.
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| CN110235862A (en) * | 2019-06-19 | 2019-09-17 | 北京林业大学 | A method of utilizing the toxicity of Drosophila melanogaster behavior stress reaction evaluation pollutant |
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| CN1969653A (en) * | 2006-11-09 | 2007-05-30 | 武汉农大生物科技有限公司 | Antivirus microbe additive for silkworm and preparation method thereof |
| CN104404125A (en) * | 2014-12-17 | 2015-03-11 | 广东省农业科学院蚕业与农产品加工研究所 | Rapid screening method for pebrine resistant germplasms |
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