CN107711892A - A kind of fungi mosquitocide and compound method - Google Patents
A kind of fungi mosquitocide and compound method Download PDFInfo
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- CN107711892A CN107711892A CN201710940310.5A CN201710940310A CN107711892A CN 107711892 A CN107711892 A CN 107711892A CN 201710940310 A CN201710940310 A CN 201710940310A CN 107711892 A CN107711892 A CN 107711892A
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- lagenidium giganteum
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- Zoology (AREA)
- Health & Medical Sciences (AREA)
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- Pest Control & Pesticides (AREA)
- Biotechnology (AREA)
- Agronomy & Crop Science (AREA)
- Plant Pathology (AREA)
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Abstract
The invention provides pythium fungi and Lagenidium giganteum to be used in combination the purposes in mosquito eradication medicine is prepared, wherein, the weight proportion of described pythium fungi and Lagenidium giganteum is：1 10 parts of pythium fungi, 1 10 parts of Lagenidium giganteum.Present invention also offers a kind of fungi mosquitocide and compound method.Pythium fungi is used in combination the present invention with Lagenidium giganteum, and effect exterminating mosquito is good, lasting period length, is significantly better than that and pythium fungi or Lagenidium giganteum is used alone, played the synergy of the two, achieved unexpected technique effect.
Description
Technical field
The invention belongs to field of biological control, and in particular to a kind of fungi mosquitocide and compound method.
Background technology
At present, control mosquito in the whole world mainly uses chemical insecticide.Chemical insecticide causes seriously to pollute to environment, breaks
The bad ecological balance, induction insect develop immunity to drugs, and force people constantly to develop new chemical insecticide, and enter pernicious follow
Ring, it is therefore desirable to develop green control mosquito means.
Biological control of insect pests be the method is free from environmental pollution using biology existing for nature or its product control insect,
The ecological balance is not destroyed, is not easy the resistance to the action of a drug for causing insect, turns into the promising approach of pests control.
Compared with agricultural pests, injurious forest-insect biological control, the biological control research of medical insect is very weak.It is showed
One of be exactly can be used for control medical insect biology very little.From the point of view of worldwide, be actually used in biology mosquito eradication only has
Bacillus thuringiensis var.israelensis de Barjac (Bacillus thuringiensis var.israelensis, Bti) and spherical gemma bar
Bacterium (Bacillus sphaericus, Bs) both bacteriums.Their mosquito eradication principle is stomach poison function.The association of bacterial spore institute
Or the toxin protein carried is taken in by mosquito larvae, is played a role in the alkaline environment of mosquito stomach, destroys the digestive tract wall of mosquito larvae
Entering its blood body cavity afterwards causes mosquito dead.But both bacteria preparations have significant limitations.First, the lasting period is short.Gemma
It can not be substantially colonized in Mosquito Breeding water body.Mud drum can be silted on the contrary wraps up in not ineffective, the Huo Zheshou by mosquito intake
Action of ultraviolet radiation and decompose.Therefore the lasting period is shorter, and particularly Bti can typically be maintained 2 weeks indoors, can only be maintained in outdoor
1 week or so.Calculate like this, a mosquito season (general to be calculated by 3 months) needs dispenser 10 times or so repeatedly.Bs lasting period is slightly
It is long, but also be difficult to reach 30 days in practical application.Second, Bs are weaker to the virulence of yellow-fever mosquito, to the medium lineae ablicantes of dengue fever
Yellow-fever mosquito does not act on.And communication media of the dengue fever in Guangdong is exactly aedes albopictus and Aedes aegypti, so in control dengue fever
Aspect, its effect are smaller.3rd, it is possible to the resistance to the action of a drug of mosquito, particularly Bs are induced, recently it was reported that it is easy to lure
Lead the resistance of mosquito.
Fungus insecticide has great advantage than germ insecticide.Most obvious of which be 1. because invasion link complexity without
Induce the resistance to the action of a drug of insect；2. the epidemic disease of insect can be caused, therefore there is the long lasting period；3. it can be invaded from insect body wall,
Can not be orally ingested even if insect can also be infected.So fungi is good insecticidal microorganism.But up to the present, fungi
Mosquito eradication has not been entered into the real practical stage.Once someone is using green muscardine fungus conidium control adult mosquito, or using green muscardine fungus and
The conidium control mosquito larvae effect of muscardine is all undesirable, it has not been convenient to promotes the use of.
Pythium fungi Pythium guiyangense (Pythium guiyangense, abbreviation Pg) is a kind of Strain of Mosquiti-Killing Fungus, and environment is fitted
Ying Xingqiang, its asexual reproductive phase zoospore can actively search mosquito larvae in water, in larva body wall encapsulated, send bud
Pipe, is stretched into the latter's body, growth and breeding, captures nutrition, destroys tissue, makes mosquito larvae dead.Its mycelia stretches out from mosquito corpse again
Body surface, release zoospore further infect new mosquito larvae, so as to cause epidemic disease in mosquito swarm, produce long-term restriction mosquito
The effect of population.Simultaneously as Pg mosquito eradication complicated mechanism, is related to many links, mosquito is not likely to produce the resistance to the action of a drug.But Pg
Mosquito eradication process it is slow, mosquitocidal effect unobvious in firm apply, thus be not easy to be received by everybody, thus its application is limited.
Pythium fungi card ground is rotten mould (Pythium carolinaria, abbreviation Pc), and a kind of Strain of Mosquiti-Killing Fungus, and it goes out
Mosquito mechanism and effect exterminating mosquito are similar with Pg.
Notification number is that the A of CN 102907458 patent discloses a kind of biological mosquitocide, by Strain of Mosquiti-Killing Fungus Pg or Pc with going out
Mosquito bacterium Bti or Bs compounding are made, but short because of Bti the and Bs lasting periods, in fact it could happen that its mosquito eradication effect is connected not with Pg/Pc
On phenomenon, cause effect exterminating mosquito to decline.
Therefore, find and the good new bio mosquitocide of development effectiveness is the task of top priority.
The content of the invention
It is an object of the invention to provide a kind of fungi mosquitocide and compound method.
The invention provides pythium fungi and Lagenidium giganteum to be used in combination the purposes in mosquito eradication medicine is prepared, wherein,
Described pythium fungi and the weight proportion of Lagenidium giganteum are：
Pythium fungi 1-10 parts, Lagenidium giganteum 1-10 parts.
The weight of two kinds of fungies is calculated with wet mycelia,
Wet mycelia：Refer to mycelia to be filtered dry, suck dry moisture with hand untill gently not water outlet is pinched.
Wherein, the weight proportion of described pythium fungi and Lagenidium giganteum is：
3 parts of pythium fungi, 3 parts of Lagenidium giganteum.
Wherein, described pythium fungi is that Pythium guiyangense and/or card ground are rotten mould；Preferably, 3 parts of Pythium guiyangense, big chain
3 parts of chytrid.
Present invention also offers a kind of fungi mosquitocide, and it is prepared by the fungi of following weight proportioning：
Pythium fungi 1-10 parts, Lagenidium giganteum 1-10 parts.
Wherein, it is prepared by the fungi of following weight proportioning：
3 parts of pythium fungi, 3 parts of Lagenidium giganteum.
Wherein, described pythium fungi is that Pythium guiyangense and/or card ground are rotten mould；Preferably, 3 parts of Pythium guiyangense, big chain
3 parts of chytrid.
Wherein, it is using pythium fungi and Lagenidium giganteum as active component, adds pharmaceutically acceptable auxiliary material or auxiliary
The preparation that helping property composition is prepared；Wherein, the formulation of the preparation is capsule, tablet, powder, pill, supensoid agent, breast
Agent.
Present invention also offers the preparation method of said medicine, and it comprises the following steps：
A, pythium fungi, Lagenidium giganteum are taken, is cultivated respectively；
B, two kinds of fungies after culture are mixed, adds pharmaceutically acceptable auxiliary material or complementary composition is prepared
Preparation.
Wherein, in step a, the pythium fungi is that Kweiyang corruption bacterium and/or card ground are rotten mould；
The culture medium of the culture is SFE, KPYG2 or rice fluid nutrient medium, and the temperature of culture is 25 DEG C ± 1 DEG C, training
The foster time is 6-7 days.
Present invention also offers purposes of the aforementioned pharmaceutical compositions in mosquito eradication medicine is prepared.
The meaning of lasting period：North temperate zone, a general mosquito season calculated by 3 months.If controlling mosquito with Bti, because
Its lasting period only has 7-10 days, and to preserve control mosquito effect, just necessary every repetition dispenser in 10 days once, is applied at least 9 times for 3 months.
If mosquitocide can be 3 months with holding effect, only use once just.Substantial amounts of manpower and materials and financial resources can be thus saved, especially
It is to carry out mosquito eradication in the huge wetland of area, the lasting period mosquitocide of length is very valuable.
Lagenidium giganteum (Lagenidium giganteum, abbreviation Lg), belongs to chain chytrid section chain Chytridium
(Lagenidium), mosquito eradication ability is strong, to being multiplied in pond, tire, trees, concrete box, garden Water-accumulating water cylinder ponding and rice field
A variety of mosquito larvaes be respectively provided with good preventive and therapeutic effect.It infects the mechanism of mosquito larvae and is similar to Pg/Pc, therefore is invaded with active
Enter mosquito host, and cause the epiphytotics effect of mosquito, and be not easy the resistance to the action of a drug for causing mosquito.
The present invention uses pythium fungi Pg or Pc, and the fungi mosquito eradication of certain proportion compounding preparation is carried out with Lagenidium giganteum Lg
Agent, have the following advantages that：
1st, mosquito eradication scope is wide, and its mosquito eradication spectrum is significantly expanded when comparing the two single use, and effect exterminating mosquito is good；
2nd, the lasting period is grown, and because Lg can be colonized in Mosquito Breeding water body, therefore has the longer lasting period.It can be with
The slow Pg/Pc work of working is used as good linking, so as to play a part of preferably controlling mosquito larvae；
3rd, Lg and Pg/Pc shows to act synergistically in the ecosystem.The two syntrophism, phase mutual supporting function.I.e.
Pg/Pc presence has to be grown beneficial to Lg in water, and the fungal colonization that they are formed in water can preferably play control mosquito
Effect.The effect exterminating mosquito of fungi mosquitocide of the present invention, which is substantially better than, is used alone pythium fungi Pg/Pc or Lagenidium giganteum Lg.
Therefore, the present invention combines pythium fungi and Lagenidium giganteum Lg, has played the synergy of the two, has taken
Obtained unexpected technique effect, there is provided a kind of mosquito eradication medicine of excellent effect.
Obviously, according to the above of the present invention, according to the ordinary technical knowledge and customary means of this area, do not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The embodiment of form by the following examples, the above of the present invention is remake further specifically
It is bright.But the scope that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following example.It is all to be based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description of the drawings
One is tested outside Fig. 1 rooms, mosquito density change is compared and (monthly counted)
Two are tested outside Fig. 2 rooms, mosquito density change is compared and (monthly counted)
Three are tested outside Fig. 3 rooms, mosquito density change is compared (based on week)
Embodiment
It is described further below with embodiment, but the present invention is not limited to these embodiments.
Reagent that the present invention uses, equipment are known product, are obtained by buying commercially available prod.
Culture medium used in the present invention is as follows：
1.KPYG2 culture mediums
(solid medium adds agar 1.5-2%)
2. sunflower seeds culture medium (SFE)
(1) take raw sunflower seeds (shell) 100g at a high speed crush 1 minute
(2) distilled water 1000ml is added to crush 2 minutes at a high speed
(3) with 4 layers of filtered through gauze
(4) distilled water 1000ml is added in filter residue, is mixed, then with 4 layers of filtered through gauze
(5) filtrate will mix twice
(6) dispense, it is quick-frozen standby
(7) used time thaws, and dilutes 1 times with distilled water
3. rice medium
(1) take raw sunflower seeds (shell) 100g at a high speed crush 1 minute
(2) distilled water 1000ml is added to crush 2 minutes at a high speed
(3) with 4 layers of filtered through gauze
(4) distilled water 1000ml is added in filter residue, is mixed, then with 4 layers of filtered through gauze
(5) filtrate will mix twice
(6) dispense, it is quick-frozen standby
(7) used time is dissolved, and the μ g of biotin 20, autoclaving are added per 100ml.
The training method of fungi is as follows in the present invention：
1. agar plate culture
Above-mentioned culture medium adds 1.5%-2% agar, is down flat plate.By mycelium inoculation on flat board, put 25 DEG C ± 1 DEG C culture.
2. Liquid Culture
By the wet mycelium inoculations of 0.5g in 100ml aforesaid liquid culture mediums, shaking table culture is put.Shaking speed 120r/min, temperature
25 DEG C ± 1 DEG C of degree.
The preparation of 1 mosquitocide of the present invention of embodiment
1st, Strain of Mosquiti-Killing Fungus Pg, Pc and Lg are cultivated 6 days using SFE fluid nutrient mediums shaking table respectively, 25 DEG C ± 1 of cultivation temperature
DEG C, shaking speed 120rpm；Filter out mycelia, sterile water washing 3 times, add water to original volume, pulverizer crushes at a high speed (Pg and Pc10
Second, Lg 6 seconds), filtering, moisture is sucked, making wet mycelia, (wet mycelia refers to mycelia to be filtered dry, and suck dry moisture is extremely gently pinched not with hand
Untill water outlet)；
2nd, by Pg and Lg 1:1-10 part by weight mixes, or Pc and Lg 1:1-10 part by weight mixes, or Pg+Pc and Lg
1:1-10 ratios mix, and obtain Pg+Lg and Pc+Lg, and tri- kinds of wet mycelia of Pg+Pc+Lg respectively, are packed under aseptic condition.
The preparation of the mosquito eradication capsule of the present invention of embodiment 2
1. Strain of Mosquiti-Killing Fungus Pg, Pc and Lg are cultivated 6 days using SFE fluid nutrient mediums shaking table respectively, 25 DEG C ± 1 of cultivation temperature
DEG C, shaking speed 120rpm；Filter out mycelia, sterile water washing 3 times, add water to original volume, pulverizer crushes at a high speed (Pg and Pc10
Second, the Lg6 seconds), it is standby；
2. prepare 1%~3% (W/V) sodium alginate soln；
3. Strain of Mosquiti-Killing Fungus mycelia suspension is pressed 1 with sodium alginate soln respectively:4 ratios mix, and mix thoroughly；
4. above-mentioned mixed liquor is slowly dropped into 1%~5% (W/V) calcium chloride solution respectively, microcapsule granule is formed, and is sunk
Form sediment, microcapsules are filtered out after 0.5~3 hour, clear water rinses standby；
5. by Pg microcapsules and Lg microcapsules 1:1-10 ratios mix, or Pc microcapsules and Lg microcapsules 1:1-10 ratios are mixed
Close, or Pg+Pc microcapsules and Lg microcapsules 1:1-10 ratios mix, and obtain Pg+Lg and Pc+Lg, and Pg+Pc+Lg tri- respectively
Product is planted, is packed under aseptic condition.
The preparation of the mosquito eradication capsule of the present invention of embodiment 3
1. cultivating Strain of Mosquiti-Killing Fungus Pg, Pc and Lg respectively 6 days using SFE, KPYG2 or rice fluid nutrient medium shaking table, cultivate
25 DEG C ± 1 DEG C of temperature, shaking speed 120rpm；Filter out mycelia, sterile water washing 3 times, add water to original volume, pulverizer high speed powder
It is broken (Pg and Pc10 seconds, Lg6 seconds), standby；
2. prepare 1%~3% (W/V) sodium alginate soln；
3. Strain of Mosquiti-Killing Fungus mycelia suspension is pressed 1 with sodium alginate soln respectively:4 ratios mix, and mix thoroughly；
4. above-mentioned mixed liquor is slowly dropped into 1%~5% (W/V) calcium chloride solution respectively, microcapsule granule is formed, and is sunk
Form sediment, microcapsules are filtered out after 0.5~3 hour, clear water rinses standby；
5. being mixed in the ratio of 1 times of amount Pg or Pc microcapsules and the 1-10 times of Lg microcapsules measured, or 1 is pressed by Pg+Pc and Lg:
1-10 ratios mix, and obtain Pg+Lg and Pc+Lg, and tri- kinds of different products of Pg+Pc+Lg respectively, are packed under aseptic condition.
Beneficial effects of the present invention are illustrated below by way of test example：
The effect measuring of 1 mosquitocide of the present invention of test example
First, outdoor manual simulation's water body method
1st, experimental method：
(1) plastics keg (bucket bottom diameter 13cm, bucket depth 22cm), running water 4L is contained, adds sterile soil 50g, added respectively
Enter fungi to be measured (mycelia suspension or with mycelia agar block), irregularly add mosquito larvae or mosquito ovum, weekly secondary sampling Detection bucket
Interior mosquito number.
Method of counting：Every barrel is fetched water 3 spoonfuls in mosquito habitat, counts mosquito number respectively by age.Compare different disposal bar
Under part, how much is the number of mosquito in each bucket.
(2) plastic barrel (bucket bottom diameter 36cm, bucket depth 80cm), depth of water 65cm, add sterile soil 1000g, and addition is treated
Bacterium (mycelia suspension or with mycelia agar block) is surveyed, irregularly adds mosquito larvae or mosquito ovum.
(3) observational technique
A observes mosquito density：Mosquito number in secondary sampling Detection bucket weekly.Method of counting：Every barrel is assembled in mosquito
3 spoonfuls of place's water intaking, mosquito number is counted by age respectively.Under the conditions of comparing different disposal, how much is the number of mosquito in each bucket.Mosquito is close
It is better to spend smaller explanation effect exterminating mosquito.
B：Observed using post mosquito：Small nylon wire is fixed on plastic foam and swum in water, takes late two age or early three ages
Mosquito larvae 25 is only put into small nylon wire, is placed in the water body of test fungi, is taken out after 48 hours, put laboratory and continue to raise,
Infection conditions are observed, to determine and compare the ability that fungi in the bucket of different disposal controls mosquito.Post mosquito infects more, says
The effect of Strain of Mosquiti-Killing Fungus is stronger in bright bucket.
2nd, experimental result
(1) mosquito swarm situation of change in keg
The outer manual simulation of 3 secondary chambers, quasi- natural environment mosquito eradication experiment have been done altogether.
Using the keg of 6 liters of capacity, interior 4 liters of running water of Sheng, sterilizing soil 50g/ buckets are added, laboratory is irregularly added and raises
Foster Culex quinquefasciatus mosquito larvae or mosquito ovum, stainless steel basket is capped on bucket, counted for 2-3 time weekly.Per 3 spoonfuls of barrelage, sum is taken.Adopt
Statistical analysis is carried out with SPSS.
Experiment one amounts to observation 54 times；Experiment two amounts to observation 29 times；Experiment three amounts to observation 23 times.
Note：Control group refers to：Mosquitocide is not added with, other conditions are identical with mosquitocide group processing.
New bio mosquitocide used in experiment one is Lg+Pg, is the agar block with mycelia, adds Pg and Lg agar respectively
Block 45cm2/ bucket (the two agar block volume is identical, and bacterium dosage is approximate quite).
New bio mosquitocide used in experiment two is Lg+Pg, is the mycelium after homogenate, adds Pg and Lg mycelia respectively
Each 20ml/ buckets of suspension (the two mycelia suspension volume is identical, and bacterium number is identical).
New bio mosquitocide used in experiment three is Lg+Pc, is the agar block with mycelia, adds Pc and Lg agar respectively
Block 45cm2/ bucket.
Wherein, mycelia suspension cultural method is as follows：Strain of Mosquiti-Killing Fungus Pg, Pc are cultivated using SFE fluid nutrient mediums shaking table respectively
With Lg 6 days, 25 DEG C ± 1 DEG C of cultivation temperature, shaking speed 120rpm；Filter out mycelia, sterile water washing 3 times, add water to substance
Product, pulverizer crush (Pg and Pc10 seconds, Lg 6 seconds) at a high speed, you can；
Mycelia agar block cultural method is as follows：Lg or Pg or Pc mycelium suspension 1ml are taken, is uniformly smeared under aseptic condition
On diameter 90mm agar medium, capping, put 25 DEG C ± 1 DEG C and cultivate 6 days.
(mycelia agar block can also be by a kind of easy method of comparison：Take about 40mm2With Lg or Pg or Pc mycelia
Agar block, 90mm agar medium center is inoculated under aseptic condition, capping, 25 DEG C ± 1 DEG C is put and cultivates 6 days).
Experimental result is shown in Fig. 1-3.
It can be seen that mosquito density can be substantially reduced using fungi mosquitocide of the present invention, there is good effect exterminating mosquito.
(2) the post mosquito infection conditions in small experiment bucket (outdoor keg method)
On November 25th, 2014 (experiment the 111st day) plus post mosquito, observation fungi field planting result, are shown in Table 1.
The keg result of table 1
Note：This experiment is that middle fungi is the agar block with mycelia, and dosage is per bacterium 45cm2/ bucket.
From table 1, the post mosquito of Lg groups is only dead 5, and can't see fungal infection, has illustrated its mosquito eradication effect
Unobvious.And in Lg and Pg or Pc compounding groups, not only post mosquito is all dead and has infected Pg or Pc, and also mosquito is simultaneously
Lg is infected, illustrates the presence due to Pg or Pc, Lg life span and the time lengthening to play a role.
Compared with Bti+Pg, Lg and Pg or Pc compounding group post mosquito are not only dead, and infect two kinds of fungies simultaneously, say
The mosquito eradication ability of bright mosquitocide is stronger.
It can be seen that being used alone, Lg is ineffective, and its lasting period is good not as Pg's and Pc, and makes when Lg and Pg or Pc coordinates
With also there is improvement Lg lasting period, serves the effect of Synergistic.
(3) outdoor drum experimental result
The outdoor various processing effect exterminating mosquitos of drum experiment first month compare (average mosquito density), average mosquito density meter
Calculate：Mosquito density in counter drum 2 times a week, method of counting are to take 3 spoonfuls with the 250ml spoon place that mosquito assembles in bucket,
Mosquito is counted, is taken the mean.The average for taking institute's total twice a week again is the density of this week mosquito.
It the results are shown in Table 2.
The drum result of table 2 (each group mosquito larvae density ratio compared with)
| Group (dosage) | First week | Second week | 3rd week | 4th week |
| Control group | 150 | 200 | 180 | 244 |
| Lg(50ml) | 10 | 8 | 10 | 18 |
| Pg(50ml) | 180 | 150 | 50 | 25 |
| Pc(50ml) | 200 | 180 | 90 | 30 |
| Lg+Pg(25ml+25ml) | 10 | 9 | 8 | 5 |
| Lg+Pc(25ml+25ml) | 9 | 8 | 10 | 5 |
Note：For fungi in drum using the mycelia suspension after homogenate, the independent dosage of every kind of fungi is 50ml/ respectively
Bucket, compounding dosage is each 25ml.
It can be seen that though Lg is rapid-action, the duration is shorter, and long-term exclusive use Lg is ineffective；And Pg or Pc works slowly,
It is poor that effect is used alone；When Lg and Pg or Pc are used cooperatively, the synergy of the two has been played, under identical dosage,
When fungi compounds, effect exterminating mosquito is much higher than the two exclusive use, achieves unexpected technique effect.
2nd, field observation
1st, experimental method
Mosquito Breeding water body adds fungi to be measured in the wild, before observation plus bacterium and plus bacterium after mosquito in different times water it is close
Degree.With spoon (250ml), 5 different parts respectively take one spoonful in water body, before and after being carried out using the number of wherein mosquito as index relatively,
And and nearby the water body of fungi is not added to be compared.Microscopy is fetched to the dead mosquito found in water, to confirm whether mosquito is applied
The fungal infection put.In water mosquito larvae number more save your breath bright Strain of Mosquiti-Killing Fungus effect it is better.
Fungi to be measured：For Lg+Pg compounding groups,
Preparation method is as follows：Strain of Mosquiti-Killing Fungus Pg and Lg are cultivated 6 days using SFE fluid nutrient mediums shaking table respectively, cultivation temperature
25 DEG C ± 1 DEG C, shaking speed 120rpm；Filter out mycelia, sterile water washing 3 times, add water to original volume, pulverizer crushes at a high speed
(Pg10 seconds, Lg 6 seconds), respectively obtains the mycelia suspension of the two；
Compare Lg by mycelia suspension volume:Pg=2:3 ratio mixing, is spread on coastal waters.Dosage is 15ml/ square meter water
Face.
2nd, experimental result
The water body mosquito density is investigated before launching this fungi mosquitocide, is sampled at random in 5 different parts, mosquito density is about
For averagely 7 every spoonful.After launching 5 days, mosquito density is decreased obviously, about 3 every spoonful.To the 13rd day of dispensing, mosquito was seldom,
One can be found once in a while.Hereafter 2 months, it there is no mosquito.At this moment nearby without mosquito in the same water body for launching fungi
Density reaches averagely 10 every spoonful.Dispensing finds a large amount of fungi-infected lethal mosquitoes after 4 months in water body.These mosquitoes
Multiplied later, but it is dead by the fungal infection in water body.Continuous observation finds the 3rd year mosquito of Second Year in the water body
Density is also significantly lower than than neighbouring water body.
It can be seen that during fungi mosquitocide actual use of the present invention, effect exterminating mosquito is good.
3rd, laboratory experiment
1st, experimental method：
(1) white plastic bowl, sterilization, 200ml running water (dispersing chlorine) overnight is contained, adds late two ages or early three age mosquitos
Larva 25, add fungi to be measured (133 × 3mm of agar block with mycelia2Three pieces, or some microlitres of mycelium homogenate), then
Add the suspension 6 of chicken liver meal 4% to drip, observation 2 times, have dead mosquito just to take out microscopy daily, record dead mosquito number and infection number, calculate dead
Die rate and infection rate.
Infection standard is to see there is mycelia in polypide under microscope, including alive and dead larva.As long as seeing mycelia, just
It is denoted as infecting.
(2) polypots, volume 2000ml. are used for indoor long-term Germicidal efficacy.The interior running water 1000ml. overnight that contains goes out on a small quantity
Bacterium mud soil.Irregularly add mosquito larvae.Observational technique：Water sampling 3 times at random, wherein mosquito larvae number is counted, takes 3 sub-samplings
With, or dead mosquito is taken out, microscopy, infection number is counted, calculates infection rate.
2nd, experimental result
The effect exterminating mosquito of (1) first Wednesday kind fungi compares
Using white plastic bowl method, fungi to be measured is the agar block 133mm with mycelia2.Three pieces of every bowl of use of Pg and Pc, Lg is every
Bowl is with one piece.
It the results are shown in Table 3.
Table 3 tests the indoor result of first week
Note：Experimental group " correction infection rate " is that total infection rate subtracts the control group death rate；Fungi is wet mycelia
It can be seen that during early stage mosquito eradication, Lg effect is preferable, and Pg, Pc effect are poor.
The effect exterminating mosquito of (2) four months various dispositions compares (average mosquito density)
Using polypots method, 4 are the results are shown in Table.
Table 4 tests the indoor result (average mosquito density) of four months
Illustrate, this experiment uses polypots.Observed data is mosquito density.
It can be seen that laboratory result is consistent with outdoor drum result.Though i.e. Lg is rapid-action, the duration is shorter, long-term single
It is solely ineffective using Lg；And Pg or Pc works slowly, it is poor that effect is used alone；When Lg and Pg or Pc are used cooperatively, play
The synergy of the two, effect exterminating mosquito greatly promote, and achieve unexpected technique effect.
(3) Lg is compared with the control mosquito effect of Pg different ratios
Because Lagenidium giganteum and the rotten mould adaptability to environment are different, growth rhythm is variant, so the two must be pressed
Certain proportioning competence exertion acts synergistically well.
Using polypots method, fungi to be measured is wet mycelia, the results are shown in Table 5.
Wet mycelia preparation method is as follows：Strain of Mosquiti-Killing Fungus Pg and Lg are cultivated 6 days using SFE fluid nutrient mediums shaking table respectively, trained
Support 25 DEG C ± 1 DEG C of temperature, shaking speed 120rpm；Mycelia is filtered out, clear water is cleaned, and is added after weighing in alms bowl.Wet mycelia with
Hand is gently pinched not water outlet and is defined.
5 different fungies of table proportioning (wet mycelia unit of weight mg) control mosquito effect compares (infection rate of mosquitoes %)
| Lg:Pg(mg) | January | 2nd month | April | June |
| 10:50 | 60 | 30 | 30 | 60 |
| 20:40 | 80 | 50 | 30 | 50 |
| 30:30 | 100 | 95 | 50 | 60 |
| 40:20 | 100 | 95 | 40 | 20 |
| 50:10 | 100 | 90 | 50 | 25 |
Note：Two kinds of fungies of infection mosquito have.The infection rate of two kinds of fungies is relevant with the fungi ratio put into.
It can be seen that the effect exterminating mosquito of Lg and PgPg different ratios is variant, wherein, Lg and Pg ratio are 30:It is short when 30
Phase, long-term infection rate of mosquitoes are all higher, illustrate that effect exterminating mosquito is optimal.Less than this ratio, short-term effect exterminating mosquito is deteriorated, and
Long-term effect exterminating mosquito also has no raising；Higher than this ratio, short-term effect exterminating mosquito is suitable, but long-term effect exterminating mosquito declines.
(4) selection of different fungi cell ages
Using the small bowl method of plastics, the effect exterminating mosquito of different cell age fungies is shown in Table 6-8.
The different cell age Lg of table 6 effect exterminating mosquito (infection rate of mosquitoes, %)
| Cell age (my god) | 3rd day | 6th day | Tenth day |
| 2 | 10 | 15 | 23 |
| 3 | 15 | 25 | 90 |
| 5 | 60 | 80 | 88 |
| 6 | 80 | 100 | 100 (second batch mosquitoes) |
| 7 | 80 | 100 | 100 (second batch mosquitoes) |
Note：1.Lg wet mycelia, 30mg/ bowls.
" 2. second batch mosquito " refers to that first mosquito infects the second batch mosquito added after death completely
The different cell age Pg of table 7 effect exterminating mosquito (infection rate of mosquitoes, %)
| Cell age (my god) | 3rd day | 6th day | Tenth day |
| 2 | 1 | 3 | 5 |
| 3 | 7 | 9 | 16 |
| 5 | 25 | 28 | 30 |
| 6 | 36 | 42 | 46 |
| 7 | 32 | 38 | 42 |
Note：The Pg agar block 133mm that carry disease germs2, three pieces every bowl.
The different cell age Pc of table 8 effect exterminating mosquito (infection rate of mosquitoes, %)
| Cell age (my god) | 3rd day | 6th day | Tenth day |
| 2 | 0 | 2 | 3 |
| 3 | 4 | 7 | 15 |
| 5 | 27 | 30 | 32 |
| 6 | 29 | 45 | 50 |
| 7 | 22 | 39 | 44 |
Note：The Pc agar block 133mm that carry disease germs2, three pieces every bowl.
It can be seen that three kinds of fungies are cell age 6-7 days (i.e. by fungal culture 6-7 days), and infection rate of mosquitoes highest, effect exterminating mosquito
It is optimal, wherein more preferably cell age is 6 days.
(5) selection of different culture media
Using the small bowl method of plastics, different culture media is shown in Table 9-11 to the effect exterminating mosquito of three kinds of fungies.
6 day age Lg of the different culture media culture of table 9 effect exterminating mosquito (infection rate of mosquitoes, %)
| Culture medium | 3rd day | 6th day | Tenth day |
| KPYG2 | 60 | 70 | 85 |
| Rice adds biotin | 80 | 89 | 92 |
| SFE | 90 | 100 | 100 (second batch mosquito larvaes) |
Note：Lg wet mycelia, 30mg/ bowls.
6 day age Pg of the different culture media culture of table 10 effect exterminating mosquito (infection rate of mosquitoes, %)
| Culture medium | 3rd day | 6th day | Tenth day |
| KPYG2 | 2 | 10 | 18 |
| Rice adds biotin | 30 | 45 | 48 |
| SFE | 38 | 48 | 49 |
Note：The Pg agar block 133mm that carry disease germs2, three pieces every bowl.
6 day age Pc of the different culture media culture of table 11 effect exterminating mosquito (infection rate of mosquitoes, %)
| Culture medium | 3rd day | 6th day | Tenth day |
| KPYG2 | 1 | 6 | 19 |
| Rice adds biotin | 28 | 35 | 39 |
| SFE | 30 | 38 | 45 |
Note：The Pc agar block 133mm that carry disease germs2, three pieces every bowl.
It can be seen that each culture medium is used equally for cultivating three kinds of fungies, wherein, optimal medium is SFE.
The preparation method screening of 2 mosquitocide of the present invention of test example
1st, when mycelium powder is broken into suspension, the dynamics of crushing and the selection of time
Pc and Pg mycelium is more elongated and mutually winds more resistance to crushing.Carried out to being crushed to various sizes of mycelia
Be further cultured for observing, it is found that after mycelia is interrupted, as long as hyphal body more than 10 microns, still can be with when condition is adapted to
Living.Finally it is set to Pg (or Pc) and uses refiner high-speed homogenization 10 seconds just.If the time is oversize, hyphal body is too small, very
Bacterium is not easy to live.
Lg mycelia is elongated unlike Pg and Pc.Its mycelia is thicker, but tends to separate into many small ball (preceding spores
Ascus), it is easier to disperse.So Lg grinding time is short, 6 seconds are crushed at a high speed just.
When the 2nd, making sodium alginate micro gel capsule, the selection of sodium alginate concentration
The sodium alginate soln for comparing various concentrations makes Pg, Pc and Lg for coming sodium alginate micro gel capsule.Tried
Sodium alginate concentration from 0.2%, 0.3%, 0.5%, 1% and 2%.
By it was found that：0.2%-0.5% concentration is too low, and the microcapsules made are not easy to shape, and moisture is easily analysed
Go out.
The sodium alginate hardness of 2% concentration is too big, and the mycelia of the inside is not easy longer performance mosquito eradication effect.
And the sodium alginate of 1% concentration is used, the ultimate density of sodium alginate is 0.8% after addition mycelium suspension, is done
Into glue bead diameter be about 350 microns, hardness is moderate, is easy to apply, and the fungi of the inside is also easy to grow.
To sum up, fungi mosquitocide of the invention has significant effect exterminating mosquito, and effect exterminating mosquito is good, lasting period length, substantially
Better than exclusive use pythium fungi or Lagenidium giganteum.The present invention combines pythium fungi and Lagenidium giganteum, plays
The synergy of the two, achieves unexpected technique effect, there is provided a kind of mosquito eradication medicine of excellent effect.
Claims (10)
1. the purposes in mosquito eradication medicine is prepared is used in combination with Lagenidium giganteum in pythium fungi, wherein, described pythium is true
The weight proportion of bacterium and Lagenidium giganteum is：
Pythium fungi 1-10 parts, Lagenidium giganteum 1-10 parts.
2. purposes according to claim 1, it is characterised in that：Described pythium fungi and the weight proportion of Lagenidium giganteum
For：
3 parts of pythium fungi, 3 parts of Lagenidium giganteum.
3. purposes according to claim 1, it is characterised in that：Described pythium fungi is Pythium guiyangense and/or card ground
It is rotten mould；Preferably, 3 parts of Pythium guiyangense, 3 parts of Lagenidium giganteum.
A kind of 4. fungi mosquitocide, it is characterised in that：It is prepared by the fungi of following weight proportioning：
Pythium fungi 1-10 parts, Lagenidium giganteum 1-10 parts.
5. medicine according to claim 4, it is characterised in that：It is prepared by the fungi of following weight proportioning：
3 parts of pythium fungi, 3 parts of Lagenidium giganteum.
6. the medicine according to claim 4 or 5, it is characterised in that：Described pythium fungi is Pythium guiyangense and/or card
Ground corruption is mould；Preferably, 3 parts of Pythium guiyangense, 3 parts of Lagenidium giganteum.
7. according to the medicine described in claim 4-6 any one, it is characterised in that：It is with pythium fungi and Lagenidium giganteum
For active component, the preparation that pharmaceutically acceptable auxiliary material or complementary composition are prepared is added；Wherein, the agent of the preparation
Type is capsule, tablet, powder, pill, supensoid agent, emulsion.
8. the preparation method of medicine described in claim 4-7 any one, it is characterised in that：It comprises the following steps：
A, pythium fungi, Lagenidium giganteum are taken, is cultivated respectively；
B, two kinds of fungies after culture are mixed, adds the system that pharmaceutically acceptable auxiliary material or complementary composition are prepared
Agent.
9. the preparation method of medicine described in claim 8, it is characterised in that：
In step a, the pythium fungi is that Kweiyang corruption bacterium and/or card ground are rotten mould；
The culture medium of the culture is SFE, KPYG2 or rice fluid nutrient medium, and the temperature of culture is 25 DEG C ± 1 DEG C, culture
Time is 6-7 days.
10. purposes of the claim 4-7 any one described pharmaceutical composition in mosquito eradication medicine is prepared.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1222190A (en) * | 1997-06-17 | 1999-07-07 | 阿格拉奎斯特公司 | Nutrient medium for increasing cell yield in fermentation |
| CN102907458A (en) * | 2012-10-28 | 2013-02-06 | 郑文定 | Biological mosquitocide and preparation method thereof |
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2017
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1222190A (en) * | 1997-06-17 | 1999-07-07 | 阿格拉奎斯特公司 | Nutrient medium for increasing cell yield in fermentation |
| CN102907458A (en) * | 2012-10-28 | 2013-02-06 | 郑文定 | Biological mosquitocide and preparation method thereof |
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