CN105594750A - Microbial insecticide and preparation method thereof - Google Patents
Microbial insecticide and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a microbial insecticide, which is a concentrated cenobium composed of 18 microbial strains classified into three types. Characterized by strong insecticidal ability, fast insecticidal speed and wide insecticidal range, the microbial strains respectively are: Metarhizium anisopliae, Paecilomyces farinosus, Phytophthora nicotianae, Streptomyces amilatus, Streptomyces culicidicus, Streptomyces griseolus subsp.hangzhouensis, Streptomyces calvus, Bacillus subtilis, Bacillus sphaericus, Bacillus laterosporus, Bacillus thuringiensis subsp.aisawai, Bacillus thuringiensis subsp.merrisoni, Bacillus thuringiensis subsp.galleriae, Bacillus thuringiensis subsp.Israelensis, Bacillus thuringiensis subsp.thuringiensis, Bacillus thuringiensis subsp.berliner, Bacillus thuringiensis subsp.dendrolimus, and Bacillus thuringiensis subsp.sotto.
Description
One, technical field:
The present invention relates to a kind of novel microbial insecticide and preparation method thereof.
Two, technical background
Since twentieth century, world population increases comparatively fast, also increasing to the total demand of food. " the petrochemistry agricultural " of people's invention, chemical fertilizer and agricultural chemicals help the mankind to obtain a large amount of food resources, have promoted the mankind's development and progress.
At present, there are 7,500,000,000 populations in the whole world, after the year two thousand fifty, will reach 9,000,000,000, approaches the peak of 10,500,000,000 populations the end of this century. So the mankind can increase considerably the requirement of food. The mankind to the dependence of " oil-agriculture " also in continuous reinforcement.
According to world's authoritative department statistics, the artificial synthetic chemical substance in the whole world has more than 1,100 ten thousand kinds at present, also constantly increases with the quantity that increases every year 1000000 tons in recent years. Cause and there are a large amount of poisonous material and harmful substances and endangering global environment, ocean and air, wide spectrum feature and the chemically compositedization technical development of chemistry noxious material, also increase the difficulty of removing, degraded, decomposition, become the procephalon of the mankind long-term hostile environment pollution disaster. Family more than 1700 of existing insecticide factory of China, produces approximately 350,000 tons of former medicines every year, process preparation 80～1,000,000 ton, and more than 40 of pesticide active ingredient kinds, approximately more than 7000 of product, but also a large amount of external highly toxic pesticide not re-used of import applies domestic soil. A large amount of pesticide producing out, in being human use, are being poisoned the mankind oneself again. Particularly most of agricultural chemicals is the toxin agricultural chemicals with interior absorption, and toxicity is directly absorbed in the histocyte of stem of plant, leaf, root, and enters fruit, Interior Seed, causes the permanence of murder by poisoning, intractable very surprising.
The use of agricultural chemicals and the relation of environmental protection; many scientists are in the infringement problem of arguing intensely about reality and potentiality; but it is apparent having a fact; use even press recommendation amount, some agricultural chemicals (particularly pesticide) really can retain considerable time and form the pollution to soil and water after use. In some instances, although working concentration is little, be also a serious threat. For example, in the food of the every ration of quail, there is the drinox of 1mg/kg also can cause 100% death.
Since half a century, people are accustomed to the use of chemical synthetic pesticide, and bring thus a well-known difficult problem: food, soil, underground water, rivers and lakes, ocean, air have all been subject to the severe contamination of chemical residual, non-target insect and other biological are produced to side effect simultaneously, caused the destruction of the ecosystem. The insect species and the quantity that chemical pesticide are produced to resistance and recovery ability constantly increase. In addition,, owing to synthesizing using and dealing with improperly of highly toxic pesticide, many non-lethal events occur again and again.
In view of many chemical synthetic pesticides are to producing many side effects, many both developed and developing countries are all constantly recognized environmental pollution that chemical synthetic pesticide causes and the harm of some toxicological effect. Thus, microorganism and natural plants, aspect Development of Novel agricultural chemicals, become the key player that people pay close attention to gradually.
Due to above various reasons, chemical noxious material, residues of pesticides toxin causes very big impact to environment for human survival, so the present invention relates to utilize microorganism to kill off the insect pests, replace chemical pesticide, nontoxic to people and animals, not welding is a kind of safe biological method.
Three, summary of the invention:
The present invention builds pesticide bacterial strain system, wherein Bacillus sphaericus (Bacillussphaericus) TS-1 and Bacillus thuringiensis H4 are two kinds of important insect pathogenic bacterias, the former is suitable with T-M-1 bacterial strain with external 1593-4 to the toxicity of Culex pipiens pallens, the latter is to armyworm, corn borers etc. have high virulence, these toxalbumin are coded by chromosome or DNA, pass through Protoplast Fusion Technique, the different breeding bacterial strains of bacillus are merged, can obtain the recombinant bacterial strain that not only kills Diptera but also kill fish lepidopterous larvae. obtain the stable fusion recombinant bacterial strain that both kills lepidopterous larvae from Dipel, obtained success.
For this reason, the invention provides a kind of microbial insecticide, be prepared from by any two classes or any 9 to the 18 kinds of bacterial classifications that are selected from following microorganism fungus kind:
Metarhizium anisopliae, paecilomyces farinograph, Phytophthora nicotianae, ring streptomycete, streptomyces culicidicus, light grey streptomycete Hangzhou subspecies, bald naked streptomycete, bacillus subtilis, Bacillus sphaericus, bacillus laterosporus, bacillus thuringiensis-Aizawa subspecies, bacillus thuringiensis-Mohs subspecies, bacillus thuringiensis-galleria mellonella waxmoth subspecies, bacillus thuringiensis-Israel subclass, bacillus thuringiensis-Su Yun gold subspecies, bacillus thuringiensis-Bai Shi subspecies, bacillus thuringiensis-pine moth subspecies, the bacillus thuringiensis-subspecies of dampinging off.
Preferred microbial insecticide of the present invention, is prepared from by following 18 kinds of bacterial classifications:
Metarhizium anisopliae, paecilomyces farinograph, Phytophthora nicotianae, ring streptomycete, streptomyces culicidicus, light grey streptomycete Hangzhou subspecies, bald naked streptomycete, bacillus subtilis, Bacillus sphaericus, bacillus laterosporus, bacillus thuringiensis-Aizawa subspecies, bacillus thuringiensis-Mohs subspecies, bacillus thuringiensis-galleria mellonella waxmoth subspecies, bacillus thuringiensis-Israel subclass, bacillus thuringiensis-Su Yun gold subspecies, bacillus thuringiensis-Bai Shi subspecies, bacillus thuringiensis-pine moth subspecies, the bacillus thuringiensis-subspecies of dampinging off.
In 18 kinds of bacterial classifications, 3 kinds of fungies, their part by weight is 1-2:1-2,4 kinds, actinomyces, and their part by weight is 1-2:1-2,11 kinds, bacterium, their part by weight is 1-2:1-2, wherein fungi, actinomyces, the part by weight of bacterium three class bacterial classifications is, fungi 45-55%, actinomyces 15-25%, bacterium 25-35%. Preferably, in 18 kinds of bacterial classifications, 3 kinds of fungies, their part by weight is 1:1,4 kinds, actinomyces, their part by weight is 1:1,11 kinds, bacterium, their part by weight is 1:1, wherein fungi, actinomyces, the part by weight of bacterium three class bacterial classifications is 50%, actinomyces 20%, bacterium 30%.
Process for preparing microbial insecticide of the present invention, is characterized in that, step is as follows:
18 kinds of bacterial classifications, 3 kinds of fungies, 4 kinds, actinomyces, 11 kinds, bacterium, respectively mixes three class bacterial classifications according to the ratio of weight ratio 1:1; Mix by the part by weight of three major types type fungi 50%, actinomyces 20%, bacterium 30% again; Mixed bacteria liquid is added in nutrient solution, put into seeding tank and cultivate, on inspection without miscellaneous bacteria, obtain liquid culture; If desired, in liquid culture, add calcium carbonate, spray dry; Obtain solid product, the batching composition of the culture medium that incubation is used is analysis for soybean powder 1.2%, dextrin 0.6%, peptone 0.3%. Dipotassium hydrogen phosphate 0.2%, calcium carbonate 0.5%, magnesium sulfate 0.4%, ammonium sulfate 0.05%, soya-bean oil 0.2%, all the other are water, nutrient solution pH value is 7.2.
Preferably, preparation method is as follows:
Add 90 kilograms after purifying, in nutrient solution, to put into seeding tank 10 kilograms of mixed bacteria liquids, at 30 DEG C ± 1 DEG C of temperature, stirring per hour 200 times, cultivates 6-8 hour; Every gram of bacteria containing amount of microscopy reaches 2,000,000,000/gram time, stop cultivating, check and be certified seed bacterium liquid without miscellaneous bacteria, qualified bacterium liquid, in 5% ratio, is added in fermentation tank; Cultivate 20-22 hour, on inspection without miscellaneous bacteria, bacterium liquid obviously becomes gemma and crystal, content 2,000,000,000/gram, be liquid culture.
Microbial insecticide of the present invention, is characterized in that, killing ability improves desinsection effect than single insecticide and reaches 51%.
Microbial insecticide of the present invention, is characterized in that, can kill chafer, mosquito, nematode, Lepidoptera, the hundreds of kind insects such as Hymenoptera and coleoptera.
18 kinds of microorganism fungus kinds of three class of the present invention are:
Three of fungies
Be respectively:
ACCC---30101 Metarhizium anisopliaes (Metarhiziumanisopliae)
ACCC---30190, paecilomyces farinograph (Paecilomycesfarinosus)
ACCC---30677, Phytophthora nicotianae (Phytophthoranicotianae)
Four, actinomyces,
Be respectively:
ACCC---41456, ring streptomycete (Streptomycesamilatus)
ACCC---41132, streptomyces culicidicus (Streptomycesculicidicus)
ACCC---40075, light grey streptomycete Hangzhou subspecies (Streptomycesgriseolussubsp.hangzhouensis)
ACCC---41451, bald naked streptomycete (Streptomycescalvus)
11, bacterium,
Be respectively:
ACCC---11088, bacillus subtilis (Bacillussubstilis)
ACCC---11096, Bacillus sphaericus (Bacillussphaericus)
ACCC---11079, bacillus laterosporus (Bacilluslaterosporus)
ACCC---10016, bacillus thuringiensis (Bacillusthuringiensissubsp.aisawai)
ACCC---10031, bacillus thuringiensis Mohs subspecies (Bacillusthuringiensissubsp.merrisoni)
ACCC---10027, bacillus thuringiensis galleria mellonella waxmoth subspecies (Bacillusthuringiensissubsp.galleriae)
ACCC---10038, bacillus thuringiensis subsp israelensis (Bacillusthuringiensissubsp.Israelensis)
ACCC---10035 bacillus thuringiensis Su Yun gold subspecies (Bacillusthuringiensissubsp.thuringiensis)
ACCC---10022, bacillus thuringiensis Bai Shi subspecies (Bacillusthuringiensissubsp.berliner)
ACCC---10062, bacillus thuringiensis pine moth subspecies (Bacillusthuringiensissubsp.dendrolimus)
ACCC---10033. Thuringiensis cladosporioides bacillus abrupt inverted sublow specie (Bacillusthuringiensissubsp.sotto)
Bacterial classification of the present invention all belongs to prior art, can buy and obtain from the market.
In the pesticide bacterial classification system that the present invention builds, have 18 bacterial classifications of three mushrooms, wherein: fungi has three, respectively insect chafer, pine tag electronics and larva are had to toxic action. Actinomyces have four, respectively mosquito and multiple lepidoptera pest are had to toxic action. Bacterium has 11, and wherein three eight of bacillus respectively have the Dipel of desinsection special efficacy, and they have respectively the various pests such as poisoning corn borer, cabbage caterpillar, mosquito, nematode, Lepidoptera and Diptera.
The feature that bacterial classification of the present invention is selected is: one selects the strong bacterial classification of insect toxic action to obtain better poisoning ability and effect. Two select to have the bacterial classification of different insect pests, to increase the broad spectrum disinfestation feature of pesticide. Three select different desinsection mushrooms, to obtain the interdependence of mushroom bacterial classification, increase spawn activity and the maintenance symbiosis effect of stronger active time.
The present invention relates to rising and insecticidal mechanism of microbial insecticide.
The microorganisms such as fungi, bacterium, virus all can make insect obtain communicable disease. The situation of bacterium, fungi and virus infection insect is comparatively common, to the research of insect disease from useful insect silkworm and honeybee. Chinese medicine Cordyceps sinensis is also by the insect bodies of fungi autoeciousness, but the understanding of insect-borne infection cause of disease is associated with microbiological development in modern age. Know that the white muscardine of silkworm is caused by fungi muscardine until begin for 1835. Shi Ao state scarab beetle (AnisoaustriacaHbst) Infective, for utilizing the early test of microbial control insect. Since 20 beginnings of the century, expand to and research viral infectious bacillary to insect. Research early has: the control of locust coccobacillus to desert locust, and the control of Japanese beetle bacillus to Japanese beetle, the control of Dipel to corn borer, and the control test of nucleopolyhedrosis virus to military poison moth and diprion hercyniae etc.
After various microbial infection insects, insect often shows the abnormality in function and behavior, the symptom (symptom) of meaning, and the constructional variation of polypide, the symptom (sign) of meaning. The two combines symptom and symptom and is generically and collectively referred to as syndrome (syn-drome) (Steinhaus and Martigoni, 1970). Conventionally be there will be the various disease conditions such as action and abnormal reaction (show as slow in action, slow in reacting etc.), gastricism (show as heavyly subtract, stop taking food, vomiting, dysentery shape etc.), maldevelopment (poor growth, can not normally cast off a skin, pupate and emergence etc.), dysgenesia (fecundity reduce, can not lay eggs etc.), body colour variation to show by the insect of infected by microbes, these symptom can be used as the foundation of differentiating that under field conditions (factors) insect catches an illness. The syndrome that cause of disease of the same race causes generally has certain feature, but also can have difference in various degree under different environmental conditions. Generally speaking, because the lethal polypide of fungal infection is withered or rigid; Die from the polypide tissue liquefaction of polyhedrosis virus, body body is easy to break; The polypide that dies from bacterial disease is softening, and body fluid is thinning, the liquid flowing out from oral cavity and anus and break the equal foul smelling of liquid that flows out; Due to the lethal polypide worm of different reasons, belong to normal enteric bacteria originally and also can breed rapidly, often exceed real lethal cause of disease, the stench also can disorganize producing and cause the confusion in diagnosis.
The pathogenetic bacteria of insect generally can be divided into the two large classes that form gemma and do not form gemma, and according to the parasitics of insect being further divided into obligate and two facultative classes. Bucher (1960) by the characteristic of pathogenetic bacteria and the required condition of causing a disease as infective dose, infect position, selectivity to host and the particular form of effect Insect Pathogenic bacterium be divided into four classes, be i.e. obligate pathogenetic bacteria, facultative spore bacteria, facultative pathogenetic bacteria and the pathogenetic bacteria that hides.
(1) obligate pathogenetic bacteria obligate pathogenetic bacteria is the extremely strong double anaerobies bacillus of a class obligate, and specific insect host is had to very strong pathogenicity. As chafer emulsus disease pathogen bacterium, it is a class obligate bacillus that chafer larva is had to pathogenicity. According to reporting for work, it is multiple that emulsus disease pathogen bacterium is isolated in Yi Cong different regions, countries in the world, wherein important with Bacillus popilliae, the gentle dead bacillus Australia mutation of slow dead bacillus, they can produce pathogenicities (Dutky, 1963) to 23 kinds of chafer larvas.
(2) the facultative spore bacteria of facultative spore bacteria refer to can produce gemma and can be on synthetic medium normal growth one bacterioid of growing, wherein have the spore bacteria Dipel monoid that can produce parasporal crystal and the bacillus that does not produce crystal. Wax bacillus is the representative species that does not produce the bacillus of crystal, be a kind of bacillus that often can find in soil, and separate in the insect bodies such as the Chang Kecong coleoptera of falling ill, Lepidoptera, Hymenoptera, infected larva is stiff, body wall is black dull, and polypide is softening to be shunk. Larva through injection treatment is often dead in 14～48h.
(3) that hides conventionally lives in insect without this bacterioid of gemma pathogenetic bacteria and obtains in alimentary canal, be mainly some asporogenic bacillus, comprise gas bacillus, the proteus etc. of pseudomonas aeruginosa, Pseudomonas fluorescens and the enterobacteriaceae of pseudomonadaceae. Seldom cause at ordinary times disease, once enter insect body cavity, can show stronger pathogenicity. These bacteriums depend mainly on some incitant by the ability that enters body cavity in intestines, cause morbidity as extreme humidity, insect eat wound that the infecting of abnormal food, other cause of diseases, parasite or cannibalism cause etc., be generally common in the insect population of artificial feeding.
(4) facultative without gemma pathogenetic bacteria as clayey Sai Shi bacillus, can have tens of kinds by parasitic insect, be Gram negative bacillus pumilis, do not produce gemma, bacterium colony be pale red or cerise on agar medium, is easy to identify also adularescent bacterial strain. Under field conditions (factors), clayey Sai Shi bacillus can be found in corn borer, black cutworm, corncob noctuid are waited for death polypide. Although this bacterium also has the feature of potential, cause that disease is comparatively common, there is the ability of certain intrusion haemocoele, therefore classify facultative cause of disease as. This bacterium is invaded after polypide haemocoele, can in 1～3d, cause that septicemia makes host's death, with the most common in the Natural Population of locust section.
In the disease of insect, infect by disease fungus the nosomycosis (mycosis) causing and account for 60%. Fungi is the eucaryote of the low grade of a class, unicellular, or the cell being broken up by majority forms, and or not containing chlorophyll, can not seek photosynthesis and Formed hydrate, without organs such as the root of differentiation, stem, leaves. The trophosome of fungi is mycelia, and most of fungies can be ruptured and breed with mycelia fragment or mycelia, and most fungies again can be with sporogenesis. Large several times to tens times of the bacterium that the fungi relevant with insect compared,
Hypha,hyphae mean breadth is 3～10 μ m. Mycelia is disagreed, have without tabula, whole mycelia be one unicellular, as Phycomycetes: what have has a tabula, is many cells, as Ascomycetes and deuteromycetes etc.
The modes of reproduction of disease fungus can be divided into two kinds of vegetative propagation and generative propagations. Vegetative propagation is to form asexual spore, is divided into again sporangiospore and conidium two classes. Most fungies without tabula produce sporangiospore, after sporangium maturation, break, and bulk storage goes out spore, and spore sprouts, grows, forms new individuality. Conidiophore can be carried and wide dispersion by wind, water and animal, is that disease fungus infects the topmost approach of insect. Generative propagation mycelia without the fungi of tabula in most generation in conjunction with spore, there are the most ascospores that produce of fungi of tabula. Most fungies have asexual and sexual two generations in the history of life; often alternate with each other, the alternation of generations of meaning, the bacterium colony of the bacterium of fungi is fine hair shape or loose flocculence conventionally; its vegetative hyphae and aerial hyphae all can form different pigments, and spore also has different colors. Some fungi of infecting insect is obligate parasite, relies on life host for its nutritional need. Having some is half parasite, also has general saprophytic bacteria. Saprophytic growing also can cause that obvious disease occurs susceptible insect under certain conditions, but the inferior natural disposition saprophytic bacteria growing on body after polypide death is easily mistaken as lethal germ.
The microorganism that the present invention selects will possess more following basic attributes:
1. virulence is high, and microbial inoculum virulence is enough to overwhelm the premunition of insect, can reliablely and stablely make insect morbidity lethal.
2. good stability, microbial inoculum not reason is subject to the impacts such as natural environment (as daylight, dry etc.), artificial treatment, suspending agent, carrier, storage, different insecticide-applying way and lowers desinsection effect.
The longevity of residure long, and have the ability of natural propagation in pest population.
(4) effect rapidly.
5. selective, and to controlling object severe toxicity, and nontoxic to plant, beneficial insect and mammal etc.
6. can large-scale production, economic security.
Process route of the present invention is to have above 18 kinds of different microbial inoculums of typical three class, first to be stored by the agriculture bacterial classification of country
Technological core of the present invention is the pesticide for general microorganism, and desinsection object is single, the feature that desinsection effect is weak. 18 kinds of strong bacterial classifications of desinsection effect of programmed screening form powerful cenobium, select three class bacterial classifications get the diversified broad spectrum activity feature of its desinsection and build.
The source and the feature that the present invention relates to 18 kinds of bacterium of three classes are as follows:
Fungi
ACCC---the AS3.3675 of clay fertilizer institute ← Institute of Microorganism, Academia Sinica of 30101 ← Chinese Academy of Agricultural Sciences ← Yulin area sand control institute, separates from paranthrene tabaniformis larva; For anti-corn borer, chafer, clearwing moth etc.
Paecilomycesfarinosus (Dicks.) Brown powder Paecilomyces varioti
ACCC---clay fertilizer institute ← Beijing Forestry University of 30190 ← Chinese Academy of Agricultural Sciences force literary composition of presenting oneself before (a monarch) is given; Original number: 8601, control dendrolimus tabulaeformis.
Bright giving wished by ACCC---tobacco research institute of 30677 ← Yunnan Province; Original number: ZML086; Locality: Yunnan; Separation source: root-knot nematode egg; Be used as biological control.
Actinomyces
ACCC---41456 ← Hubei Province biological pesticide Engineering Research Center; Original number: HBERC00797; Metabolite has insecticidal activity; Separation source: soil; Separate place: five li of rivers of Lushi, henan.
ACCC---raw institute is planted in institute of microbiology ← Shanghai, Guangzhou, 41132 ← Guangdong Province; Original number: GIM4.12MIK4.1; Kill mosquito larvae; Separation source: soil; Separate place: Guangzhou.
StreptomycesgriseolussubsphangzhouensisACCC---40075 ← Zhejiang Agriculture academy of sciences; Original number: S26; Product kills aphid element, very strong to aphid, red spider and some lepidopterous larvae toxic action.
ACCC---41451 ← Hubei Province biological pesticide Engineering Research Center; Original number: HBERC00743; Metabolite has insecticidal activity; Separation source: soil; Separate place: Zhejiang dragon trip.
Bacterium
ACCC---11088 ← Institute of Microorganism, Academia Sinica; AS1.0933; Control peanut nematodiasis.
ACCC---11096 ← Institute of Microorganism, Academia Sinica ← U.S.;=AS1.1672; Original number: 2115; Kill mosquito.
ACCC---the AS1.0864 of clay fertilizer institute ← Microbe Inst., Chinese Academy of Sciences of 11079 ← the Chinese Academy of Agricultural Sciences; Energy kill mosquitoes larva; Produce lysozyme.
Bacillusthuringienissubsp.aisawaiACCC---10016 ← Wuhan microbial pesticide factory; Original number: O96; Prepare antigen reference culture, poisoning lepidopterous insects.
Bacillusthuringienissubsp.merrisoniACCC---10031 ← Wuhan microbial pesticide factory; Original number: O12; Poisoning lepidopterous insects.
ACCC---10027 ← Microbe Inst., Chinese Academy of Sciences; Poisoning cabbage caterpillar.
ACCC---10038 ← Chengdu Hua Hong industrial corporation ← Taian Shandong biological study institute ← World Health Organization (WHO); Kill mosquitoes larva, wriggler effect are better.
Bacillusthuringienissubsp.thuringiensACCC---10035 ← China Agriculture Academe Fertilizer Institute; Original number: D25; Antiphagin poisoning corn borer; Separation source: natural seed selection from HD-187 bacterial strain and obtaining.
ACCC---10022 ← China Agriculture Academe Fertilizer Institute; Original number: D26; Control lepidopterous insects; Separation source: bacterium powder.
ACCC---10062 ← Soviet Union; Poisoning pine moth.
Bacillusthuringienissubsp.sottoACCC---10033 ← Wuhan microbial pesticide factory ← Britain; Original coding: 016; Prepare antigen reference culture, poisoning lepidopterous insects.
Four, specific implementation method
By following specific embodiment, the present invention is further illustrated, but not as restriction of the present invention.
Example one: the present invention relates to a kind of microbial insecticide agent and process for producing same process route is:
The first step. The cultivation of bacterial classification: according to the title of bacterial classification and numbering, buy 18 kinds of bacterial classifications of three major types (sand pipe bacterial classification) back by Chinese microbial preservation committee agricultural microorganism center; Under aseptic condition, sand pipe bacterial classification is moved and received in the slant tube that beef extract, peptone agar medium are housed; Observe its purity, without plaque, have 95% above form normal without miscellaneous bacteria, smear for microscopic examination; Normal bacterial classification good purity is carried out to single thalline liquid shaking table to be cultivated; Quantity expands shaking table facility scale or repeats preparation once on request, meets the requirement of next step cultivation.
Second step. The expansion of bacterium liquid is cultivated: first 18 pure liquid of bacterial classification are mixed in the ratio of 1:1; Mix by the part by weight of three major types type fungi 50%, actinomyces 20%, bacterium 30% again; 10 kilograms of mixed bacteria liquids are added to 90 kilograms after purifying in liquid medium, and batching composition is analysis for soybean powder 1.2%, dextrin 0.6%, peptone 0.3%. Dipotassium hydrogen phosphate 0.2%, calcium carbonate 0.5%, magnesium sulfate 0.4%, ammonium sulfate 0.05%, soya-bean oil 0.2%, in the nutrient solution that pH value is 7.2; Put into seeding tank, at 30 DEG C ± 1 DEG C of temperature, stirring per hour 200 times, cultivates 6-8 hour; Every gram of bacteria containing amount of microscopy reaches 2,000,000,000/gram time, stop cultivating, check and be certified seed bacterium liquid without miscellaneous bacteria. Qualified bacterium liquid, in 5% ratio, is added (formula as above) in fermentation tank; Cultivate 20-22 hour, on inspection without miscellaneous bacteria, bacterium liquid obviously becomes gemma and crystal, and content reaches 2,000,000,000/gram, can be finished product fermentate.
The 3rd step. The preparation of solid fungicide: first give the calcium carbonate that adds 8-10 in finished product bacterium liquid, stir 30 minutes; Pass through plate-frame filtering; By adding calcium carbonate quantity 10% to add spreader-sticker, stirring 30 minutes, furnishing pulpous state; Stamp again head tank, spray dry; Spray dried bacterium powder through being pulverized and mixed evenly, take a sample to check qualified; Seal with plastic bag packaging in time; Putting into shady and cool dry storehouse preserves.
Example two, three quasi-microorganisms that the present invention relates to are fungi, actinomyces and bacterium.
Fungi has three, is respectively:
ACCC---the AS3.3675 of clay fertilizer institute ← Institute of Microorganism, Academia Sinica of 30101 ← Chinese Academy of Agricultural Sciences ← Yulin area sand control institute, separates from paranthrene tabaniformis larva; For anti-corn borer, chafer, clearwing moth etc.
Paecilomycesfarinosus (Dicks.) Brown powder Paecilomyces varioti
ACCC---clay fertilizer institute ← Beijing Forestry University of 30190 ← Chinese Academy of Agricultural Sciences force literary composition of presenting oneself before (a monarch) is given; Original number: 8601, control dendrolimus tabulaeformis.
Bright giving wished by ACCC---tobacco research institute of 30677 ← Yunnan Province; Original number: ZML086; Locality: Yunnan; Separation source: root-knot nematode egg; Be used as biological control research.
Four, actinomyces, are respectively:
ACCC---41456 ← Hubei Province biological pesticide Engineering Research Center; Original number: HBERC00797; Metabolite has insecticidal activity; Separation source: soil; Separate place: five li of rivers of Lushi, henan.
ACCC---raw institute is planted in institute of microbiology ← Shanghai, Guangzhou, 41132 ← Guangdong Province; Original number: GIM4.12MIK4.1; Kill mosquito larvae; Separation source: soil; Separate place: Guangzhou.
StreptomycesgriseolussubsphangzhouensisACCC---40075 ← Zhejiang Agriculture academy of sciences; Original number: S26; Product kills aphid element, very strong to aphid, red spider and some lepidopterous larvae toxic action.
ACCC---41451 ← Hubei Province biological pesticide Engineering Research Center; Original number: HBERC00743; Metabolite has insecticidal activity; Separation source: soil; Separate place: Zhejiang dragon trip.
11, bacterium, is respectively:
ACCC---11088 ← Institute of Microorganism, Academia Sinica; AS1.0933; Control peanut nematodiasis.
ACCC---11096 ← Institute of Microorganism, Academia Sinica ← U.S.;=AS1.1672; Original number: 2115; Kill mosquito.
ACCC---the AS1.0864 of clay fertilizer institute ← Microbe Inst., Chinese Academy of Sciences of 11079 ← the Chinese Academy of Agricultural Sciences; Energy kill mosquitoes larva; Produce lysozyme.
Bacillusthuringienissubsp.aisawaiACCC---10016 ← Wuhan microbial pesticide factory; Original number: O96; Prepare antigen reference culture, poisoning lepidopterous insects.
Bacillusthuringienissubsp.merrisoniACCC---10031 ← Wuhan microbial pesticide factory; Original number: O12; Poisoning lepidopterous insects.
ACCC---10027 ← Microbe Inst., Chinese Academy of Sciences; Poisoning cabbage caterpillar.
ACCC---10038 ← Chengdu Hua Hong industrial corporation ← Taian Shandong biological study institute ← World Health Organization (WHO); Kill mosquitoes larva, wriggler effect are better.
Bacillusthuringienissubsp.thuringiensACCC---10035 ← China Agriculture Academe Fertilizer Institute; Original number: D25; Antiphagin poisoning corn borer; Separation source: natural seed selection from HD-187 bacterial strain and obtaining.
ACCC---10022 ← China Agriculture Academe Fertilizer Institute; Original number: D26; Control lepidopterous insects; Separation source: bacterium powder.
ACCC---10062 ← Soviet Union; Poisoning pine moth.
Bacillusthuringienissubsp.sottoACCC---10033 ← Wuhan microbial pesticide factory ← Britain; Original coding: 016; Prepare antigen reference culture, poisoning lepidopterous insects.
Formula is that 18 kinds of bacterium mix for the first time with 1:1 ratio. Carry out secondary mixing by three class bacterium liquid again, secondary mixed proportion is that fungi accounts for 50%, actinomyces 20%, and bacterium accounts for 30%.
Example two: the present invention is sprayed on the finished product of preparation on pest body epidermis, the imbibition of bacterium spore, approximately can sprout and grow 1-2 germinations through 8-12 hours, secretion chitin lyase, invade to sprout germ tube, extend for mycelia later, directly absorb mixed polypide liquid nutrient and arrive and fill the air in polypide blood, hinder blood circulation. The metabolite Oxalates of mycelia is accumulation in a large number in blood, and basicity declines, and loses original transparency and stagnant muddiness causes the change of physicochemical property, makes metabolism disorderly and dead.
Example three. By finished product preparation of the present invention, be sprayed on plant, enter enteron aisle through insect population, 48 hours enteron aisle miospore germination rates 37%. In 60-72 hours, mycelia is invaded intestinal walls, and makes digestive juice enter haemocoele, and within 84 hours, digestive juice pH value is down to 5.0 by 7.0, and mycelia stretches out from polypide coria, valve, sense organ hole, and the death rate reaches 95%.
Example four: by finished product preparation of the present invention, be sprinkled upon polypide upper, fungi is poisonous to insect, but not direct kill insects, and destroyed polypide epidermis, be conducive to insecticidal bacteria invade, cause syndrome, do not treat the mycotic slow generating process of onset, and sick and dead rapidly by bacterium system. Utilize with regard to the present invention that the innovative idea of multiclass bacterium mixed insecticidal is correct and preparation as insecticiding effect is remarkable.
Example five: microbial insecticide of the present invention and market single culture pesticide are to pakchoi field cabbage caterpillar poisoning experiment. Spray pesticide of the present invention after five days, group 32 blue or green worms in trial zone have 25 to occur pathology or death; Group 31 cabbage caterpillars in check plot have 12 pathology or death occur. Prove that this pesticide is higher by 51% than contrast insecticidal effect.
Claims (8)
1. a microbial insecticide, is characterized in that, by any two classes that are selected from following microorganism fungus kindOr any 9 to 18 kinds of bacterial classifications are prepared from:
Metarhizium anisopliae, paecilomyces farinograph, Phytophthora nicotianae, ring streptomycete, streptomyces culicidicus, light grey strepto-Bacterium Hangzhou subspecies, bald naked streptomycete, bacillus subtilis, Bacillus sphaericus, bacillus laterosporus, Su YunGold bacillus-Aizawa subspecies, bacillus thuringiensis-Mohs subspecies, bacillus thuringiensis-galleria mellonella waxmoth subspecies,Bacillus thuringiensis-Israel subclass, bacillus thuringiensis-Su Yun gold subspecies, bacillus thuringiensis-Bai Shi subspecies, bacillus thuringiensis-pine moth subspecies, the bacillus thuringiensis-subspecies of dampinging off.
2. microbial insecticide according to claim 1, is characterized in that, by following 18 kinds of bacterial classificationsBe prepared from:
Metarhizium anisopliae, paecilomyces farinograph, Phytophthora nicotianae, ring streptomycete, streptomyces culicidicus, light grey strepto-Bacterium Hangzhou subspecies, bald naked streptomycete, bacillus subtilis, Bacillus sphaericus, bacillus laterosporus, Su YunGold bacillus-Aizawa subspecies, bacillus thuringiensis-Mohs subspecies, bacillus thuringiensis-galleria mellonella waxmoth subspecies,Bacillus thuringiensis-Israel subclass, bacillus thuringiensis-Su Yun gold subspecies, bacillus thuringiensis-Bai Shi subspecies, bacillus thuringiensis-pine moth subspecies, the bacillus thuringiensis-subspecies of dampinging off.
3. microbial insecticide according to claim 2, is characterized in that, in 18 kinds of bacterial classifications, and fungi3 kinds, their part by weight is 1-2:1-2,4 kinds, actinomyces, and their part by weight is 1-2:1-2, thin11 kinds of bacterium, their part by weight is 1-2:1-2, wherein fungi, actinomyces, the weight of bacterium three class bacterial classificationsRatio is, fungi 45-55%, actinomyces 15-25%, bacterium 25-35%.
4. microbial insecticide according to claim 1, is characterized in that, in 18 kinds of bacterial classifications, and fungi3 kinds, their part by weight is 1:1,4 kinds, actinomyces, and their part by weight is 1:1, bacterium 11Kind, their part by weight is 1:1, wherein fungi, and actinomyces, the part by weight of bacterium three class bacterial classifications is50%, actinomyces 20%, bacterium 30%.
5. process for preparing microbial insecticide claimed in claim 4, is characterized in that, step is as follows:
18 kinds of bacterial classifications, 3 kinds of fungies, 4 kinds, actinomyces, 11 kinds, bacterium, by three class bacterial classifications respectively according to weight ratioThe ratio of 1:1 is mixed; Press again the weight of three major types type fungi 50%, actinomyces 20%, bacterium 30%Ratio is mixed; Mixed bacteria liquid is added in nutrient solution, put into seeding tank and cultivate, on inspection without miscellaneous bacteria,Obtain liquid culture; If desired, in liquid culture, add calcium carbonate, spray dry; Obtain solidProduct, the batching composition of the culture medium that incubation is used is analysis for soybean powder 1.2%, dextrin 0.6%, peptone0.3%. Dipotassium hydrogen phosphate 0.2%, calcium carbonate 0.5%, magnesium sulfate 0.4%, ammonium sulfate 0.05%, soya-bean oil0.2%, all the other are water, and nutrient solution pH value is 7.2.
6. preparation method according to claim 5, is characterized in that, step is as follows:
Add 90 kilograms after purifying, in nutrient solution, to put into seeding tank 10 kilograms of mixed bacteria liquids,At 30 DEG C+1 DEG C of temperature, stirring per hour 200 times, cultivates 6-8 hour; Every gram of bacteria containing amount of microscopy reaches2000000000/gram time, stop cultivating, check and be certified seed bacterium liquid without miscellaneous bacteria, by qualified bacterium liquid by 5%Ratio, adds in fermentation tank; Cultivate 20-22 hour, on inspection without miscellaneous bacteria, bacterium liquid obviously becomes gemma and crystal,Content 2,000,000,000/gram, be liquid culture.
7. microbial insecticide according to claim 1, is characterized in that, killing ability is killed than singleWorm agent improves desinsection effect and reaches 51%.
8. microbial insecticide according to claim 1, is characterized in that, can kill chafer, mosquitoWorm, nematode, Lepidoptera, the hundreds of kind insects such as Hymenoptera and coleoptera.
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| CN113142247A (en) * | 2021-01-23 | 2021-07-23 | 山东九一生物科技股份有限公司 | Preparation method of mosquito killing preparation special for livestock and poultry breeding colony house |
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| CN113142247A (en) * | 2021-01-23 | 2021-07-23 | 山东九一生物科技股份有限公司 | Preparation method of mosquito killing preparation special for livestock and poultry breeding colony house |
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Application publication date: 20160525 |