CN1095758A - Storing fungal cultures and conidial method - Google Patents
Storing fungal cultures and conidial method Download PDFInfo
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- CN1095758A CN1095758A CN 93116796 CN93116796A CN1095758A CN 1095758 A CN1095758 A CN 1095758A CN 93116796 CN93116796 CN 93116796 CN 93116796 A CN93116796 A CN 93116796A CN 1095758 A CN1095758 A CN 1095758A
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Abstract
Describe the packing green muscardine fungus and belonged to fungal cultures or conidial method.In one embodiment, in the insect infection cell that attracts insect, provide fungal cultures, then with the fungi infestation of lethal dose it, wherein packing keeps in the cell high humidity being arranged, allow free exchanging gas, and can not see through the microorganism that comprises fungal spore, virus and bacterium.In second embodiment, under low relative humidity and oxygen level condition, pack fungus conidium.Can reactivate the conidium that is used to control insects such as cockroach, fly, ant, soft-bodied insects, grassland insect and caterpillar then.
Description
Storage that the present invention relates to improve and reactivate insect pathogenic fungus, particularly green muscardine fungus belong to (Metarhizium) fungal cultures and conidial tool and method.
The application is the David W.Miller that submitted on May 27th, 1992, and the name of Paul Perry and CarolAnn Johnson is called the part continuation application of the u.s. patent application serial number 07/889,594 of " preservation condition of insect infection chamber ".
There are many kinds of insects can cause serious rural economy loss, and can between people and other faunas, spread disease.Recently, used different chemical insecticides to control these insects.But unfortunately, the sterilant price is very high and can constantly work the mischief to environment, but particularly this hazardness long-term existence.In addition, also might produce the resistance to insecticides insect, so that need to use a large amount of sterilants also to need handle once more with different sterilants.Use chemical insecticide also can cause the injures and deaths of non-target insect.
Can use high toxicity chemical insecticide commonly used insect pathogens in addition to control harmful insect.Can use bacterium such as Bacillus thuringiensis to be sprayed at insect such as gypsymoth are infected on the responsive plant, and obtain certain success.Fungi is the another kind of insect pathogens likely that is suitable for as the biotic pesticide of control insect.Modal growth and the modes of reproduction of fungi are vegetalitas or vegetative propagation, comprise sporulation, are spore-germination then.Asexual spore comprises conidium, and the top of sporogenous cell and both sides form the mycelial filamentary texture of many cells on mycelia.In control environment, conidium germinates, and becomes big, and produces germ tube.Germ tube is grown or mycelia behind the certain hour, forms mycelium then.When by contacting with the propagulum alive of insect pathogenic fungus when inoculating insect, the parasitism of setting up simultaneously between fungi and target insect promptly takes place to infect.Along with the development of course of infection, fungi is kill insects gradually just.
United States Patent (USP) 5,057,316 and 5,057, No. 315 control is disclosed and elimination comprises
The adult of flying insect such as Lian, housefly and other insects such as corn rootworm is in the method for interior insect.By infecting the chamber, can pathogenic fungi infestation insect be arranged to insect when using in order to sufficiently high concentration, thus the purpose that reaches the control insect and destroy the insects.Remain with the fungal spore that insect is had the work of virulence in the cell, prevent that fungi is subjected to the environment destruction of (comprising rain, UV-light and wind), or as the decoy of insect so that with a large amount of spore inoculating insects.Fungal cultures can provide spore continuously in long-time.Spore is attached on the insect, and has germ tube to penetrate in the insect body, thereby makes insect dead in 3 to 4 days.The design of cell can make it to become unique attractive substance of insect as its shape and color.In addition, food or bait also can be used to improve the magnetism of cell to insect.Though main the infection is by the outside contact, also can come infected insect by the insect spore that contacts with each other or ingest.In some cases, the fungus conidium of being ingested also is virose.
The historic restriction that insect pathogenic fungus is obtained business success is that they lack good preservation characteristics.Though proved United States Patent (USP) 5,057,316 and 5,057, cell described in No. 315 all is effectively under laboratory and real field test conditions, but they must stand long-distance transporting, and keeps stable during standing storage at room temperature and maybe can become commodity alive.
Two kinds of entomopathogenicity fungies the most desirable are green muscardine fungus (Metarhizium aniso-pliae) and muscardine (Beauveria bassiana).Then, because the restriction that is run in fungus breeding body such as conidial stabilization, storage and storage back reactivate, the commercialization of these two kinds of fungi products has up to now only obtained very limited success.
Include 6 stages conidial life history: formation, ripe, ripe back, activation, dormancy and germination.In the dormant stage, along with the prolongation of quiescent period, spore is in the physiologically active state of continuous reduction, and external environment condition is had maximum tolerance.Two types dormancy is arranged: the structure dormancy comprise can not be simply inherent mandatory dormancy by providing the condition that is suitable for growing to be overcome; The exogen dormancy is bestowed by environment, stops dormancy when the condition that occurs being suitable for growing.Can be by not germinate specific nutrition required plain or exist inhibitor to suppress the exogen dormancy, and can under some envrionment conditions, keep.
Recognize the comparable fresh preservation longer time of exsiccant microbial spore, cell and other type propaguluies before this.But most of scientists accept such viewpoint, i.e. viable cell dehydration can cause the integral body protuberance of film, irreversibly lose integrity (the Cro ω e﹠amp of 26S Proteasome Structure and Function; Cro ω e, Stabilization of Membranes in Anhydro-biotic Orga nisms, Membranes, Metabolism and Dry Organism-s, A..C.Leopold, Comstock Publishing Co., Ithica, NY, pp.188-209(1986); Beker﹠amp; Rapoport, Conservation of Yeast by Dehydra-tion, Advances in Biochemical Engineering/Biotechnology, Vol.35, Biotechnology Methods, ed.A.Fiechper, Springer Verlag, pp.127-171(1987).This viewpoint has obtained that germination speed obviously reduces this true support when spore or other types microbial reproduction soma are dry.Relevantly cause that by dehydration most of research work of structural damage concentrate on (Beker﹠amp on the yeast; Rapoport, 1987).Filamentous fungus can produce dissimilar propaguluies, and conidium then is a kind of propagulum that can produce in a large number at short notice.But conidium all can not tolerate dehydration.Finding that the conidium of various insect pathogenic fungus is very sensitive to drying treatment, and can very fast its viability of forfeiture (No. 92100010.5, people's such as Eyal european patent application).Made a lot of effort (Australian patent application AU-8-81286/87 of people such as Andersch for exploitation mycelium preparation; No. 92100019.5, european patent application), but because the mycelium preparation is unstable under high-temperature, and limited their application.
With regard to storing temp and relative humidity (R.H.) some researchs (Bell, 1975 have been carried out in the usefulness influence of conidium stability and green muscardine fungus and muscardine; Clerk﹠amp; Madelin, 1965; Daoust﹠amp; Roberts, Effect of formulation on the Viabili-ty of Metarhiziun anisopliae Conidia, J.of Invertebrate Patholo-gy, Vol.41, pp.151-160(1983); Ka ω akami, 1960; Ka ω akami﹠amp; Kikuni, 1965; Steinhaus, 1960; Walstad, J..D., " Effects of Envi-ronmental Conditions on T ω o Species of Muscardine Fungi(Beauveria bassiana and Metarhizium anisopliae) ", J.of Inver-tebrate Pathology, Vol.16,221-226(1970)).The result of these researchs shows, (survival time of 26 ℃ ± 97%R.H. or 19 ℃+97%R.H.) is the longest under the moderate temperature that high R.H. is arranged for the conidium of green muscardine fungus.But can lose its germinating power at short notice in 37 ℃ of following conidiums.Germination is first stage that grows up in the insect pathogenic fungus.
During transportation and warehouse storage, temperature can be up to 37 ℃.Prior art is not scrutinized the conidial state that does not germinate.Quicken and the technology that causes the green muscardine fungus conidium to germinate simultaneously though used fresh conidium investigation of materials, also need in distilled water, flood 10-44 hour, and suitable nutrition sources (Dillon﹠amp is provided; Charn-ley, 1985).The conidium of insect pathogenic fungus such as green muscardine fungus and muscardine is natural hydrophobic.When they are suspended in water, there is high surface tension on the interface between water and conidium surface, stop conidium to absorb the necessary water that germinates.By the different tensio-active agent of scrutiny, might relax this problem.The feature of tensio-active agent is that its molecular structure can be divided into distinct portions in different knowing on the degree.A part is hydrophilic, and another is hydrophobic.The method that is used for the hydrophilic-hydrophobic matter of quantitative tensio-active agent is hydrophile-lipophilic thing balance (HLB) method.In this method, HLB numerical value is allocated in each tensio-active agent, and is associated with application surface promoting agent suitably by conversion.Design proportion is so that more hydrophilic tensio-active agent has higher HLB numerical value.Be used for chemical insecticide preparation though will screen the HLB method of tensio-active agent, the HLB method that will be used to screen suitable tensio-active agent be applied to dry and store after reactivate microbial reproduction body promptly be a new field.In addition, about O when under differing temps-R.H. condition concurrent, storing conidium
2Level influences the conidium viability and knows little.It is crucial that oxygen germinates for fungal spore.
Therefore, an object of the present invention is to provide the Method and kit for of a kind of shelf-life of the pathogenic epiphyte that is increased on the nutritional medium, particularly green muscardine fungus.
Another object of the present invention provides the packing of the insect infection cell that is used for pathogenic epiphyte of improvement.
Another order of the present invention provide under comparatively high temps, store the entomopathogenicity fungi conidium to increase the method for its shelf-life.
A further object of the present invention reactivates conidial usability methods after providing storage.
The present invention introduced packing contain fungal cultures or entomopathogenicity fungi particularly green muscardine fungus belong to the method for the conidial infection cell of fungi.These methods can be under up to 37 ℃ temperature the standing storage conidium, and can have continue after make it to reactivate with control cockroach, fly, ant, soft-bodied insects, grassland insect, insects such as caterpillar, and do not have or seldom lose fungi infestation and cause the ability of sensitive insect death.
Studies have shown that the conidium of green muscardine fungus has good viability when being to preserve under atmosphericoxygen content (20%O2) and room temperature and high relative humidity (90-100%R.H.) condition, but at 37 ℃ of following other R.H. and the next forfeiture survival ability of oxygen level condition.At low oxygen-containing water flat (0-5%O2), under the 1-10%R.H. level conditions, have respectively 56% and 26% conidium can 25 ℃ and 37 ℃ store 2 months down after germination.When the conidium with green muscardine fungus is immersed in suitable surfactant mixture, be in 10 the ethoxylated alcohol time as hydrophile-lipophilic thing balance (HLB) number, the percentage of germination under 25 ℃ and 37 ℃ is increased to 77% and be increased to 74% from 26% from 56% respectively.With control wax moth, the set bioassay method test of (Galleria mellonella) larva of big wax stores bimestrial conidium under three kinds of varying environment conditions.Condition of storage is respectively: (1) 25 ℃, and 90-100%R.H. and atmosphericoxygen level; (2) 25 ℃, 0-10%R.H. and 0-5%O
2; (3) 37 ℃, 0-10%R.H. and 0-5%O
2The percentage demonstration of the larva alives of handling with these three groups different conidiums, the conidium of storage and fresh conidium are similar or better.Under low relative humidity, pack, provide an environment that dewaters rapidly and present dormant state to conidium.The conidium of dehydration is stored in and makes it in the low-oxygen environment to keep dormancy between the shelf lives, thereby still can survive at elevated temperatures.Yet the surface tension that causes along with dehydration increases, and has reduced percentage of germination after storage.Use suitable tensio-active agent then to reduce conidial surface tension and help rehydration and interrupt dormancy.
Therefore, contain in the preferred embodiment of infection cell of conidium in packing, packing is with air-locked, waterproof steam, the aluminium foil of flexible bag material such as aluminium foil and low density polyethylene ene coatings is made.Packing can guarantee to have thick-and-thin oxygen and R.H. level between the shelf lives.Can use siccative to produce the low R.H. environment of 0-10%.Can pack certain and can add oxygen absorbent in each pouch to cause low-oxygen environment with siccative.Available moisture agar block keeps 90-100%R.H..Can in the packing that contains the 35-40%R.H. air, seal conidium so that the R.H. of 35-40% to be provided.
Can be with required oxygen and R.H. condition packing conidium in for example infecting the such device of cell.Also can pack conidium under proper condition in enormous quantities, be suspended in then in the mixing tank of suitable spraying use with kill pests.In this embodiment, be expected to comprise the capillary tensio-active agent that is used to reduce fungus conidium, to help the survival rate that it germinates and don't influences fungus conidium in using carrier.With conidium with use carrier and mix after, can use conventional equipment and method this Fungicide for preparing to be sprayed on the plant of desiring to carry out insect control and elsewhere.
Contain in the preferred embodiment of infection cell of fungal cultures in packing, the flexible bag material that is provided is waterproof steam and water, and packs fungal cultures under high humidity.
Fig. 1 α shows that four batches of green muscardine funguss are in the percentage survival rate that is infecting under uncontrolled condition after being incubated different time (fate) in the cell.
Fig. 1 b shows the percentage survival rate of the cockroach contact with the infection cell that contains fungi, and wherein fungi is aerobic (black diamonds-) or anoxic (O-) preserve under the condition different time (my god).Not the survival rate of the cockroach that contacts with fungi be denoted as (empty square-.
Fig. 2 is the three-dimensional plot that infects the survival rate of the fungi that is incubated in the cell under different relative humidity: the percentage survival rate is mapped to the time to percentage relative humidity.
Fig. 3 diagram show contact different time (day) the percentage survival rate of back cockroach (Blatella germanica), wherein fungi is stored in 22 ℃ and the black square of 0%R.H.(respectively), the black circle of 33%R.H.(with the infection cell that contains the green muscardine fungus culture), 100%R.H.(deceives rhombus) and contrast R.H.(sky circle) under the condition.
Fig. 4 a, 4b and 4c diagram show green muscardine fungus bacterial strain ESF53(Fig. 4 a), green muscardine fungus bacterial strain ESF1(Fig. 4 b) and muscardine bacterial strain ESF2(Fig. 4 c) at 22 ℃ (black squares), 27 ℃ (empty squares), 32 ℃ (black rhombuses) and 37 ℃ (empty rhombus) number of surviving of the percentage behind the storage different time (week) down.
Fig. 5 is with respect to the packaging system that causes different conditions of storage, and green muscardine fungus is at the relation curve of storage 1 under 25 ℃ and 37 ℃ and the percentage percentage of germination after 2 months.Packaging system is single with paper tinsel, paper tinsel+Drierite
TM, paper tinsel+water agar, paper tinsel+Ageless
TM, paper tinsel+Drierite
TM+ Ageless
TM, paper tinsel+water agar+Ageless
TMCondition (from left to right) is: 25 ℃, and 40%R.H.+20%O
2; 37 ℃, 40%R.H.+20%O
2; 25 ℃, 0%R.H.+20%O
2; 37 ℃, 0%R.H.+20%O
2; 25 ℃, 40%R.H.+0%O
2; 37 ℃, 40%R.H.+0%O
2; 25 ℃, 0%R.H.+0%O
2; 37 ℃, 0%R.H.+0%O
2; 25 ℃, 100%R.H.+0%O
2And 37 ℃, 100%R.H.+0%O
2
Fig. 6 is the relation curve of the conidial percentage percentage of germination of green muscardine fungus to packaging system, and said packaging system causes different condition of storage with 37 ℃ in following 2 months at 25 ℃, wherein be determined at store the back with or handle without tensio-active agent.Packaging system is: single with paillon foil, paper tinsel+Drierite
TM, paper tinsel+water agar, paper tinsel+Ageless
TM, paper tinsel+Drierite
TM+ Ageless
TM, paper tinsel+water agar+Ageless
TMUsed tensio-active agent is the tridecanol of ethoxylation.Condition of storage is with described in Fig. 5.
Fig. 7 shows the wax moth that the conidium be used in the green muscardine fungus that stores under the bimestrial varying environment condition is handled, the big wax residing external conditions of (Galleria mellonella) larva of setting.Conidium is that (1) is fresh; (2) the water agar block is packed with maintenance 100%R.H., and is stored under 25 ℃, and (3) use Drierite
TM+ Ageless
TMPacking, and be stored under 25 ℃ or (4) 37 ℃.To on its body, there be conidial larva of new formation to be accredited as the larva that conidium forms.The mortality ratio of all treatment group is 100%.
The invention describes to storing entomopathogenic fungi, the particularly required Packaging Method of green muscardine fungus Pseudomonas and conidium thereof.Use these methods to be able to the standing storage fungus conidium, and continue after be used to control insects such as cockroach, fly, ant, soft-bodied insects, grassland insect and caterpillar.
In the business development of biology Controlling System, a fundamental is that preparation must have the rational shelf-life.Be used to guarantee that the packaging system of reasonable shelf-life must be economical, and be easy to assembling, transportation and storage.Packing must keep required fungus breeding body such as conidium to survive in the rational temperature scope and virulence is arranged.
In keeping viability, it is relative humidity (R.H.), oxygen and temperature that performance has the factor of maximum importance.Said herein " low " R.H. is meant R.H. less than 10%, or is 0-10%; " height " R.H. is meant R.H. greater than 90%, or is 90-100%.Moderate R.H. is meant 35-40%.The oxygen level generally is in the atmosphericoxygen content range, promptly 20%; The hypoxemia level is 0-5%; The hyperoxia level is 90-100%.
The condition of storage of fungal cultures
United States Patent (USP) 5,057,315 and 5,057, the entomopathogenic fungi of using as comprise green muscardine fungus and muscardine has been described, control and eliminating for No. 316
The method of Lian and other insects.Use by close by insect entry and contain insect by the pathogenic fungi pollution cell that the cell of culture forms of living, pending environment is used fungi.The geometrical shape of this device is insect to be entered behind the cell promptly with the culture of the pathomycete of effective dose contact.Find, very poor no matter the structure of cell or storage vessel how, is used in the interior insect storage power under room temperature and oxygen free condition of this insect infection cell.Do not obtaining continuously under the situation of oxygen, fungi is lost its viability, causes cell lethal infection insect effectively, though fungi might be reduced to conidial 1% survival rate and remain effective.Fungi also needs high humidity.When storing temp is 22-25 ℃ when being increased to 42 ℃ from room temperature, promptly need to increase relative humidity.
Can provide enough oxygen and high humidity in order to adapt to these requirements, to have designed, and the wrapping material of the insect of fungal cultures storage cell, such material can keep most of water vapors, allows to comprise the gaseous interchange of oxygen and carbon dioxide simultaneously.Material therefor preferably can see through and comprise fungal spore, and bacterium and virus are at interior microbe.
Now as follows to the conditional statement of shelf-life of being prolonged green muscardine fungus.These conditions reach by packing under atmosphere and 100%R.H..Selected wrapping material can make gas reach balance, can keep 100%R.H. simultaneously.
Wrapping material
The various materials that have the feature of qualification with regard to its barrier all are commercially available; That is, they can keep and make it different gas and move by layer of material.But these materials can hardly obtain with two kinds of specifications flexure.Can make the bag of the pliable and tough barrier membranes of various character and structure at an easy rate.
The most desirable material that is used for the bag cell is the low density and the high density polyethylene(HDPE) (referring to polymkeric substance ramose degree) of 2 to 8 mil thick that can seal.Other materials comprises the polypropylene that low air permeability is arranged than polyethylene.As follows, these commercially available material have been characterised in that low transmission of water vapor ability and high gas permeability.
The character of table 1 fungal cultures wrapping material
The material water vapor transmission
aGas permeability
bWater absorbs
O
2N
2CO
2
Polyethylene (low density) 1.3 550 180 2900 is low
Polyethylene (high-density) 0.3 600 70 4500 is low
Polypropylene 0.7 240 60 800 is low
A. at 95 °F, lose gram number/24 hour/100 square inches/mil under the 90%R.H. condition;
B. at 77 °F, CC number/24 under the 50%R.H. condition hour/100 square inches/mil; ASTM D1434-63.
Available commercially available other materials replaces polyethylene and polypropylene, in order to equivalent gas and water vapor transmission ability to be provided.These materials can derive from D﹠amp; B Plastics, Fair-mouth, MN or other plastic films supplier.
The condition of storage of fungus conidium
Fungal spore, conidium are not 'inertia', but will consume the oxygen of q.s.But the amount much less that fungal colony consumed of the amount specific activity of the oxygen that is consumed growth.Under high R.H. condition, conidium can not get oxygen will lose viability.But in 25 ℃, the conidium of packing under high R.H. and the atmosphericoxygen content condition is that 37 ℃ of following storages will lose its germinating power after 2 months.Studies have shown that now if direct environment is (0-5%) that does not almost have oxygen, then conidium can be at low R.H.(less than 10%) keep it to have vigor under the condition.In order to adapt to the requirement of hypoxemia and low relative humidity, can use not the wrapping material that see through any gas or water vapor and share siccative, and can when using the compound that removes deoxidation or method, use it.
Be used to exhaust or remove the material of deoxidation
There is multiple oxygen consumption agent (as oxygen absorbent) to pack with conidium.The oxygen absorbent that is used for removing oxygen in bag or any other type of container must be avirulent to fungus conidium.For example can use Cor-poration, Food division " B ", 520Madison Avenue, New York, the Ageless of N.Y.10022 available from Mitsubishi International
TMAgeless
TMMain component be Powdered activated ferric oxide, it can become ferriferous oxide and oxyhydroxide after absorbing oxygen.In gas tight container, Ageless
TMCan make oxygen be reduced to 0.01%(100ppm) or still less.Selected oxygen absorbent also must be compatible with siccative.Z type Ageless
TMBe particularly suitable for using, and can use with siccative with dry substance.The capacity that oxygen absorbent is removed oxygen in the encloses container is another factor that should consider.One bag of Ageless
TM-Z300 has the capacity that absorbs 300ml oxygen, and this is equivalent to volume of air 1500ml.
In addition, can when sealing, fill nitrogen and from packing, remove deoxidation by vacuum take-off or in sealing forward direction packing.
Be used to exhaust or the material of dry-off moisture
Available various siccative is as available from W.A.Hammond Drierite Company, and P.O.Box 60, Xenia, the Drierite of Ohio 5385
TM(anhydrous calciumsulphate), dry-off moisture from packing.Drierite
TMBe a kind of suitable siccative that can be used for the microbial insecticide packaging system,, can not discharge any moisture content that is absorbed because be exposed to high room temperature condition following time when it.Water is securely held in the calcium sulfate of half hydrated form; As therefrom removing the temperature that moisture content then need surpass 350 (177 ℃).For transport microniological proudcts under variable weather condition, this is an important feature.For dry gas, Drierite
TMHave 10-14%(weight) water capacity.A Drierite
TMDesiccant bag can or still less make in the time humidity in the ambient of sealing be reduced to-100 (73 ℃) dew points at about 10 hours.24cm * about the 200-250cm of 14cm volume
3Container use a bag.Also can use silica gel, other compounds such as some clay, polyacrylic acid derivative and other siccative cause low R.H. environment.
Wrapping material
Opposite with the material that is used for packing fungal cultures, it should be air-locked being used to pack conidial material.Being used to pack conidial preferred material is can be from Laminated Foil and Packaging, and the thickness that Portsmouth, N.H. buy is at least 0.003 inch, best 0.007 inch polyethylene-aluminium foil-polyethylene.
Also can use the oxygen transfer rate less than 0.005cc/100 square inch/sky, transmission of water vapor speed is lower than the other materials in 0.005cc/100 square inch/sky.Here, the material that will have these transport propertys is regarded " not seeing through " water and gas as.
The character of table 2 packing conidial polyethylene-aluminium foil-polyethylene bag
Thickness oxygen permeability moisture vapor permeability
O
2CC/100 square inch/g/m
2/ 24 hours
24 hours
0.003 " or 3 mils 0.005 0
0.005 " or 5 mils 0.000 0
0.007 " or 7 mils 0.000 0
Available commercially available analog material substitutes the flaggy paper tinsel, so that equivalent gas and moisture vapor permeability to be provided.Can obtain these materials by suppliers.
Pack fungal cultures to be stored and conidial method
Use ordinary method well known by persons skilled in the art such as heat sealing method with fungal cultures or conidium is close exists in the bag material.Can use the ultrasonic sealing method, but this not a preferable methods.As much as possible conidium is encapsulated in the bag of the minimum that can hold them, and other air or water are joined.
In a preferred embodiment, the conidium that is contained in the gas impermeable material can be packed with the siccative and the oxygen absorbent of q.s, to cause less than 10%R.H. in bag and to be less than 5%O
2Condition.In addition, one useful but be not in the embodiment preferred, the conidium that is contained in the gas impermeable material packed with limited amount siccative, and do not pack oxygen absorbent, in bag, to produce condition less than 10%R.H..
This packing can comprise a plurality of compartments and be used for the tolerant instrument that mixes within each space.For example, for formation is applied to the fungus breeding liquid suspension of pending region, first compartment can contain tensio-active agent, and second compartment contains the fungus breeding body.Similarly, for forming dried fungus breeding body formulation, first compartment can contain the dry surface promoting agent, and second compartment contains the fungus breeding body.
Tensio-active agent
Usable surface promoting agent (also being called wetting agent) reactivates the conidium after the storage.Tensio-active agent can be divided into five big classes: (1) is anionic, and (2) are cationic, and (3) are non-ionic, and (4) amphoteric and (5) are non-water-soluble.In most of the cases, preferred anionic and nonionic surface active agent.Various nonionic surface active agent are all specified a HLB numerical value in yardstick, wherein more hydrophilic tensio-active agent has higher HLB numerical value.The HLB method is applicable to nonionic surface active agent mostly, as HLB is 10 I-conol TDA, i.e. the tridecanol of ethoxylation (BASF Corporation, Persippany, N.J.07054), as if it can make the conidium of the green muscardine fungus of storage be able to best reactivate.Can be with Iconol TDA6(HLB=11) and Iconol TDA3(HLB=8) by 2: mixed 1(W/W) is 10 to reach HLB.For this purpose, also can be used alone or as a mixture HLB between 8-13, other nonionic surface active agent with different chemical structures, as the alcohol of segmented copolymer, ethoxylation, the lipid acid of the alkylphenol of ethoxylation, the amine of ethoxylation and/or acid amides, ethoxylation, the aliphatic ester of ethoxylation and oil (animal and plant), aliphatic ester; Lipid acid, alcohol or alkylphenol, sorbitan derivatives, sucrose and the glucose ester and the derivative of glyceryl alcohol, glycol ester, lanolin radical derivative, direactive glyceride and derivative, polymkeric substance (polysaccharide, vinylformic acid, acrylamide), propoxylation and ethoxylation.Also can use some negatively charged ion and the cationic surfactant of HLB between 8-14.In anionic or other classifications, also there is some to be suitable for the conidial tensio-active agent of reactivate, as the salt of sulfo-succinic acid salt and tartrate (Igepon T-77) as the salt of two octyl sodium sulfosuccinates, sulfonic acid such as dodecylbenzene sulfonate, naphthene sulfonic acid.The example of these tensio-active agents comprises: block polymer such as Pluronic 25R4; The alcohol of ethoxycarbonylization such as Tergitol 15 S-5, the alkylphenol of ethoxylation such as Triton N-57, the amine of ethoxylation and acid amides such as Ethomid 0-17, the lipid acid of ethoxylation such as PEG oleic acid, the lipid acid of ethoxylation and oil are as Alkamuls EL-L20, glyceryl ester such as Emerest 2421, glycol ester such as Ethox DO-9, poly-polysaccharide, poly vinylformic acid and acrylamide such as APG325 Glycoside, the lipid acid of propoxylation and ethoxylation, alcohol or alkylphenol, sorbitan derivatives such as Span60, and sucrose and glucose ester and derivative such as Crodesta F-50.Examples of anionic surfactants comprises the salt (as the dioctyl sulfosuccinate) of sulfo-succinic acid, salt (as the salt of Witco 1298 Soft Acid, the sodium salt of alkyl benzene sulphonate (ABS)), phosphoric acid acid and the tartrate of sulfonic acid.
For green muscardine fungus, the HLB number of tensio-active agent is preferably in the 9-11 scope.For muscardine, the HLB of tensio-active agent is preferably in the 9-14 scope.
According to the difference of used tensio-active agent, for the amount of one or more required mixed surfactants of reactivate conidium can be low to moderate conidial 0.01%, and high to 100%(W/W) (being that proportional range was from 1: 1000 to 100: 1).When the screening tensio-active agent, preferably carefully understand the HLB value, because in case determined just also can in the reactivate conidium, use other different tensio-active agents that have the harmless same HLB numerical value of respective type fungus breeding body, bacterial cell or other microorganisms by a certain appropriate HLB numerical value.
In a preferred embodiment, conidium is packaged in the siccative that enough produces low R.H. amount in packing, and enough in packing, causes in the paillon foil of oxygen absorbent of low-oxygen environment amount.Then with the contents mixed of this packing, with reactivate conidium before application with second packing that contains surfactant mixture or mixture suspension.In addition, preferred surfactants can be mixed with conidium and some inert material in case of necessity, as last preparation and storage as stated above.Can with afterwards with tensio-active agent blended conidium, or the material that contains conidium and tensio-active agent for preparing is made sprays, pulvis, tablet, granule or gel uses.For example, can pack the composition of conidium and these two kinds of last preparations of tensio-active agent respectively, and before application, mix it.The Fungicide for preparing fully can be made sprays, liquid medicine or pulvis then and use, with antagonism cockroach, housefly, aphid, termite or ant.
Now further set forth the present invention by the non-limiting example of the importance of the various features of following proof present method and wrapping material.
Use following material and method to determine of the influence of various conditions of storage in the following example to the viability and the effectiveness of fungal cultures.
Viability:
Use Solanum tuberosum dextrosum gel (Potato Dextrose Agar, PDA) the dull and stereotyped conidial percentage survival rate of following program determination green muscardine fungus (germination) of pressing.
Make fresh, aseptic PDA flat board.Tip with little aseptic painting brush contacts the conidium lawn gently to collect conidium.Make the brush top touch the inside of aseptic plate to remove too much conidium.Only need conidium in a small amount.Conidium is carefully also brushed on four/part of PDA flat board lightly, use the green muscardine fungus that derives from the source, different areas to repeat to brush other 3/4ths parts.Per quart brush one or two brushes get final product.Plate is incubated 11-13 hour down in 28 ℃.Be incubated after 13 hours, under compound microscope, amplify the surface of checking each inoculation zone in 200 times.Find and to check single conidial visual field.At least conidial number of counting 200 conidiums and record survival and not surviving.The survival conidium has a length at least as the germ tube of conidium diameter.Count survival conidium/total mitogenetic spore count of each 1/4th inoculation part, ask the mean value of four countings and multiply by 100 to determine the conidial percentage ratio of survival.
Render a service:
In embodiment 1 to 6, use blatta germanica (Blatella germanica) to determine to render a service with footwear shape box bioassay method (Shoe box bioassay).
Material:
Polystyrene footwear shape Storage Box (12.5 * 6.75 * 3.6 inches) has pore at the top of being stamped polyester mosquito mesh screen.
Adult Germany cockroach (blatta germanica; JK-Consulting, Amherst MA), raises the #5001 with Purina Lab Chow(Purina, Purina Mills, Inc., St.Louis MO), can in vitro freely obtain distilled water what clog with fabric.
In the environmental chamber that has temperature and humidity (128 ℃ and 75%R.H.) system and continuous data recorder, put into footwear shape box.
Method:
Vertical side with thin layer vaseline coating footwear shape box.In each footwear shape box, add autoclaved Purina Lab Cho ω agglomerate and water pipe.Put into 20 cockroaches in each box.The footwear shape box that is placed with cockroach is placed environmental chamber.Respectively put one as United States Patent (USP) 5,057 in the footwear shape box of four cockroaches, 315 and 5,057, No. 316 described infection cells.Organize in contrast with other four footwear shape boxes.
Footwear shape box is placed 28 ± 3 ℃ and 75 ± 15%R.H. environment and the insulation down of 10 hour photoperiod.Write down the cockroach mortality ratio weekly, observed for 6 weeks altogether.Do not see that when stabbing insect insect motion thinks dead with blunt.
Embodiment 1: when the forfeiture situation of cell fungi surviving power when protectorate is placed under room temperature and uncontrolled humidity
Four batches of different fungi cells are put into box under room temp and humidity.With take a sample to check the at interval viability of fungi of a few days.
The result shows shown in Fig. 1 a, and when when any long period stores not protectedly, fungi loses its viability.
Embodiment 2:
When under oxygen free condition, storing cell to the influence of fungi surviving.
Infecting cells with 5 is placed on and a series ofly is furnished with in airtight cover/quart ceramic pot.Cell in contrast is exposed to atmospheric environment, and cover is unclamped.Be exposed to the interior Ageless of introducing of cell of oxygen free condition
TM(Mitsubishi) oxygen scavenqer bag, and with the cover jam-pack.Ageless
TMBag is made up of in small, broken bits, unoxidized iron filler, begins oxidation when the latter contacts with air.When this process occurs in the environment of sealing such as these jars, promptly removed all oxygen and caused oxygen-free environment.
Data presentation shown in table 3 and Fig. 1 b effectiveness that stores the average survival rate after 42 days under these conditions and infect cell.The result proves, for existing for standing storage fungi successfully needs oxygen under room temperature and normal atmospheric humidity.
Table 3: the survival rate that infects fungi in the cell
The percentage survival rate
Control group 72.06 in 14 days
Ageless 69.94
Control group 81.54 in 42 days
Ageless 7.00
Embodiment 3: determine at the suitable wrapping material that allow fungal cultures under the oxygen flow situation
The test different materials, determine infecting the how oxygen consumed of fungi of living in the cell, and when the packing fungi, the Different Package material how because of permission oxygen by maybe not slowing down the requirement that oxygen is infeeded.
Carry out two researchs:
1) there is the cell of fungi to put into 6: (a) Rubkermaid
TMContainer (7.5 * 7.5 * 2.75 inches), (b) 4 mils (0.004 inch) new LDPE (film grade) bags (LDPE) (D﹠amp; B Plastics, Fairmouth, MN) or (c) 8 mil LDPE bags.Two duplicate samples are wherein respectively arranged, be called " A " and " B ".Though Rubbermaid
TMContainer is made with thick polypropylene hypoxemia permeable material, shifts but can oxygen take place around the container cover notch portion.As shown in table 1, the feature of LDPE mainly is its low moisture vapor permeability and its hyperoxia and carbonic acid gas permeability.
Table 4: infect cell condition in the packing after 30 ℃ store 14 days
%O
2%CO
2The % survival rate
Rubbermaid TM A 16.0 5.5 95.0
B 11.4 10.4 92.2
4 mil LDPE A 12.5 2.2 86.3
B 12.3 2.3 86.1
8 mil LDPE A 5.9 3.8 93.0
B 5.0 4.0 94.1
2) in several aluminium foil laminate bags, respectively seal 20 cells (Laminated Foil and Packaging, Portsmouth, NH).Laminated product is polyethylene-aluminium foil (0.0007 inch)-polyethylene; These wrapping material are counted as not saturating fully any gas or steam.Reservoir bag and at the oxygen and carbon dioxide content that detects thereafter wherein.Each sampling spot is all surveyed two bags of multiple.
Result as shown in table 5 proof has consumed all oxygen and enrichment carbonic acid gas.Determine the average survival rate of fungi then.
Table 5: the condition after three months in the paper tinsel bag
%O
2%CO
2The % survival rate
22℃ A 0.3 23.4 15.0
30℃ A 0 23.7 0.0
These two result of experiment have proved the degree of fungi oxygen consumed; And combine with other embodiment and to see, also proof provides the importance of proper packing.The fungi that obtains oxygen is still alive.
Embodiment 4: high humidity is to the vital role of the stability in storage of fungi
There is the infection cell of fungi to put into a series of Rubbermaid with 5
TMIn the container, to determine that relative humidity (R.H.) is to the influence of the extended storage stability of fungi in the container.The inside of a container series has been put into wet sponge and has been kept 100%R.H. to guarantee container, has put into saturated magnesium chloride (MgCl in another container series
2) solution to be to keep 30%R.H..A large amount of silica gel are then put into to keep 0%R.H. in last container series inside.All containers all are stored under the room temperature, and regularly infect take a sample to check the cell fungi surviving rate and the efficient of cell after time expand in container.
The results are shown among table 6 and Fig. 2.These results are presented at the average survival rate that storage recorded during 14 months with form and graphic form.Infect the efficient of cell when also showing 9 months in the table 3.
The result proves, is standing storage fungi successfully, and 100%R.H. is better than other humidity significantly.
Table 6: the percentage survival rate under each relative humidity condition
Relative humidity
1 week 81 61.1 69.1
2 weeks 69 74 51
3 weeks 68.5 73.1 19.2
1 month 67.3 30.5 12.7
6 weeks 58.4 11.4 0.54
2 months 53.5 28 23.9
3 months 78.3 15.3 29.8
6 months 82.33 0.08 0.74
9 months 80.54 2.33 0.75
14 months 84.29 00
Embodiment 5: more different commodity packaging materials provide the ability of suitable standing storage condition aspect for fungi.
In two series bags, respectively put into 20 and infect cell.A bag series is 8 mil LDPE.Another series is as embodiment 3 described paper tinsel laminated products.Bag is stored in 22 ℃ or 30 ℃.Provide a bag interior required 100%R.H. by the mycobiotic agar growth medium of staying behind the fungi development in the cell.Bag taking and from the series that different conditions of storage are arranged regularly as the sample of measuring gas content.Each sample is all used two bags of multiple (A and B).The gained result conforms to the data of acquisition among the embodiment 3; That is, do not keep oxygen in the paper tinsel lamination bag in a short time, but the oxygen of a great deal of is then arranged in the LDPE bag.Open bag then, fungi surviving rate of taking a sample to check the cell in it.The result shows, in the time of 3 months, and oxygen flow (LDPE) bag and the clear and definite difference of appearance between oxygen flow (paper tinsel) bag not.
Can draw such conclusion, promptly fungal cultures needs oxygen, and the LDPE bag can carry out the viability of enough gaseous interchange with long-term maintenance fungi.
Table is the condition in the paper tinsel bag in the time of 7A:3 month
%O
2%CO
2The % survival rate
22℃ A 0.3 23.4 15.00
30℃ A 0 23.7 0.00
Table is the condition in the LDPE bag in the time of 7B:3 month
%O
2%CO
2The % survival rate
22℃ A 8.5 3.0 92.1
B 8.8 2.5 90.9
30℃ A 8.7 3.0 86.0
B 4.5 4.2 87.8
Embodiment 6: relatively terms of packing is to the influence of different fungi surviving rates
Fig. 4 a, three kinds of different fungies that b and c proof stores under embodiment 3 described conditions are in the percentage survival rate of different time.Bacterial strain ESF2 is the muscardine bacterial strain, and bacterial strain ESF1 and ESF53 are the green muscardine fungus bacterial strains.
Under similarity condition, use the same wrapping material of preserving the green muscardine fungus bacterial strain, the muscardine bacterial strain can not be survived well.These results show, just are packaged in the efficient of promoting in the fungi surviving, can not be extended to fungi based on the data of packing other types microorganism.
Embodiment 7: determine the influence of various conditions of storage to the viability and the effectiveness of fungus conidium
Use following material and method to determine of the influence of various conditions of storage to fungus conidium viability and effectiveness.
The conidium cut that will store in each bag is at above-mentioned potato dextrose agar (Potato Dexztrose Agar) (PDA) on the flat board, to detect muscardine or the conidial survival rate of green muscardine fungus.With the conidia of beauveria bassiana on the PDA flat board in 22 ℃ of insulations 18 hours, and with the conidium of green muscardine fungus in 25 ℃ of insulations 15 hours.After the insulation, count the conidium number that germinates on each PDA flat board.Respectively duplicating the survival rate of bag endoconidium represents with the percentage percentage of germination.Then according to the average percentage percentage of germination of each treatment group of numerical evaluation of four copy group, and provide the standard deviation of numerical value.
Use ESC-1 bacterial strain of green muscardine fungus and the ESC-170 bacterial strain of muscardine, and used fungus breeding body type is the conidium that remains in the little weighing pan.This type is made up of the conidium of having brushed the mycelium layer and be placed in the weighing pan.Use Ageless
TMProduce direct hypoxemia (＜5%) environment.Use Z-300 type Ageless
TMBe because it is particularly suitable for drying material and can uses with siccative.1 bag of Ageless
TM-Z300 has the capacity that absorbs 300ml oxygen, and this is equivalent to volume of air 1500ml.The volume of paper tinsel bag is about 250ml.At experimental session, by producing in the water agar plate that does not have fungi and not keeping high R.H. in some bag with water vapor that conidium directly contacts.Use Drierite
TMPacking into need provide in the bag of low R.H. environment.In such paper tinsel bag, there is not gaseous interchange.
All treatment group all were incubated for 4 or 8 weeks down at 25 ℃ and 37 ℃.Each treatment group all repeats 4 parts, and the result represents with mean number plus-minus standard deviation.
Show with green muscardine fungus at atmosphericoxygen level (20%O among Fig. 5
2) result that records under the condition.The conidium of green muscardine fungus is littler than the conidium stability of muscardine.Though they have good viability when packing under 37 ℃ of normal atmospheric oxygen levels and 90-100%R.H. condition, in 37 ℃ other R.H. and the next meeting forfeiture of oxygen level conditions viability.Behind bag under 0-5% oxygen and the 0-10%R.H. condition, after 25 ℃ and 37 ℃ store 2 months, there is 56% and 26% conidium to germinate respectively.
Embodiment 8: the conidial tensio-active agent of dried green muscardine fungus of determining to be applicable to the reactivate storage
It is in the tensio-active agent that the influence screening of percentage percentage of germination is had the HLB numerical value of different chemical feature.
Surfactant soln/the suspension that 100ml is had certain HLB numerical value or chemical feature is added in the 250ml beaker.0.01g to 0.5g exsiccant conidial powder is sprinkled upon on the surface of surfactant soln or suspension.In case the conidium sample is wetting in surfactant soln, is about to solution and transfers in the 100ml graduated cylinder and put upside down (in 1 minute 30 times) repeatedly to mix it.Make conidium at room temperature sink to the bottom of graduated cylinder.Supernatant discarded is also got sedimentary conidium sample with aseptic cotton swab.The PDA flat board that will contain each conidium sample is then checked the conidial number that has germinateed in per 100 total mitogenetic spores in 25 ℃ of insulations 25 hours.
The results are shown among table 8 and Fig. 6.Fig. 6 is that the conidial percentage percentage of germination of green muscardine fungus causes relation curve between the packaging system of various conditions of storage in following 2 months 25 ℃ and 37 ℃ relatively, wherein percentage of germination after storage with or handle and determine without tensio-active agent.Packaging system is single with paper tinsel, paper tinsel+Drierite
TM, paper tinsel+water agar-agar, paper tinsel+Ageless
TM, paper tinsel+Drierite
TM+ Ageless
TM, paper tinsel+water agar+Ageless
TMUsed tensio-active agent is that hydrophile one parent refers to that thing balance (HLB) number is 10 ethoxylation tridecanol.With 10 seconds kinds of conidium rotation and in this tensio-active agent dipping 15 minutes.
Table 8: at the HLB number is 10 0.05%(W/V) in the ethoxylation tridecanol solution dipping conidium to the dry conidial influence of the green muscardine fungus that reactivates storage
Handle % percentage of germination standard deviation
Dry conidium contrasts 20.6 2.7
Rotate with Iconol TDA
The dry conidium 88.2 2.8 in 10 seconds
Rotate with Iconol TDA
Also add 15 minutes 10 seconds
The dry conidium 85.8 5.3 of dipping
Rotate with Iconol TDA
Also add 6 hours 10 seconds
The dry conidium 84.7 3.5 of dipping
The result clearly illustrates that, when being in 10 the ethoxylation tridecanol during dipping green muscardine fungus at suitable tensio-active agent and hydrophilic-lipophilic thing balance (HLB) number, percentage of germination is increased to 77% and be increased to 74% from 26% from 56% respectively.
Embodiment 9: conidial effectiveness of determining storage with bioassay method
The set effectiveness of (Galle-ria mellonella) of the conidium control wax moth that test stores in bioassay method, big wax.The last instar larvae of wax moth is by Northern Bait, 1520Knapp St., and P.O.Box 216, Chetek, Wisconsin 54728 supplies with.
Be immersed in wax moth end instar larvae in the conidial suspension that stirs in advance in small beaker and kept for 15 seconds.In Tea-leaf filtering net, handle 10 one group insect together, and be placed in the 100mm plastics plate of the wetting vermiculite of useful 1ml distilled water.After 4 days and 11 days, write down mortality ratio.All insects are all put a week again and write down external conidium and form situation.
With the conidium control wax moth that bioassay method test stores under three kinds of varying environment conditions, the set ability of (Galleria mellonella) larva of big wax.Three kinds of conditions of storage are: (1) 25 ℃, and 90-100%R.H. and atmosphericoxygen level; (2) 25 ℃, 0%R.H. and 0-5% oxygen; (3) 37 ℃, 0%R.H. and 0-5% oxygen, the storage time is 2 months.
The percentage ratio that has shown the larva of the external conidium formation of green muscardine fungus that useful these three groups different conidiums are handled among Fig. 7 proves that they and fresh conidium are similar or better.
The relation of table green muscardine fungus survival rate and the interior gas composition of paper tinsel bag and relative temperature (R.H.) after 9:2 month
Gas composition stores after two months gas composition and survival rate when sealing
%O
2%CO
2The % survival rate
25℃
R.H. 0-10%+20% O
220.9 0.0 0.0
R.H. 0-10%+0% O
20.4 0.0 56.1
R.H. 35-40%+20% O
220.2 0.5 20.1
R.H. 35-40%+0% O
20.4 0.0 15.2
R.H. 90-100%+20% O
219.3 1.2 95.0
R.H. 90-100%+0% O
20.5 0.0 37.1
37℃
R.H. 0-10%+20% O
220.5 0.0 0.0
R.H. 0-10%+0% O
20.4 0.0 26.4
R.H. 35-40%+20% O
220.4 0.3 0.0
R.H. 35-40%+0% O
20.4 0.0 0.0
R.H. 90-100%+20% O
216.7 3.3 0.0
R.H. 90-100%+0% O
20.4 0.0 0.0
In a word, low R.H. is packaged as conidium an environment that dewaters rapidly and guarantee to enter dormant state is provided.The conidium of the dehydration that stores under low-oxygen environment remained on resting stage between the shelf lives, even so they still can survive well at elevated temperatures.Yet, increase because of dehydration makes it surface tension, and after storing, reduced percentage of germination.Use suitable tensio-active agent can reduce conidial surface tension and help rehydrated and interrupt dormancy.
In this preferred embodiment, packing is with airtight, but the bag material of waterproof steam and flexure such as aluminium foil and be surrounded by that the new LDPE (film grade) of aluminium foil makes.Packing can guarantee to have between the shelf lives invariable oxygen and R.H. level.Under hypoxemia and 0-10%R.H. condition, the conidium of muscardine can survive 2 months at 25 ℃ and 37 ℃.But the percentage of germination of the green muscardine fungus that (25 ℃ and 37 ℃, hypoxemia and 0-10%R.H.) store under similarity condition has only 56% and 26% respectively.But is 10 0.05%(W/V with these conidiums at tensio-active agent such as HLB) dipping 15 minutes in the ethoxylation tridecanol, can make percentage of germination be increased to 77% and 74% respectively significantly.
To the present invention starting of packing that the method for entomopathogenic fungi and material do with change, will be conspicuous according to top detailed description to those skilled in the art.These changes and change also should be included in the present invention and to await the reply in the claim scope.
Claims (31)
1, stores the method for green muscardine fungus culture over a long time, be included under the 100% relative humidity condition in the material of airtight, waterproof steam and seal fungal cultures.
2, according to the process of claim 1 wherein that material is flexible film.
3, according to the method for claim 2, wherein material is selected from new LDPE (film grade), high density polyethylene(HDPE) and polypropylene, and wherein material thickness is between 2 to 8 mils.
4, according to the process of claim 1 wherein that material is the heat-sealing bag around the nutritional medium that contains fungal cultures.
5, according to the process of claim 1 wherein that fungal cultures is included in the infection cell of insect.
6, according to the process of claim 1 wherein that fungi is green muscardine fungus (Metarhiz-ium anisopliae).
7, the green muscardine fungus of packing belongs to fungal cultures, comprises
The wrapping material of the fungal cultures of the work on nutritional medium and parcel fungal cultures, wherein material is to see through O
2And CO
2But permeate water steam not, and the relative humidity of the fungal cultures that is wrapped is 100%.
8, according to the culture of claim 7, wherein material is flexible film.
9, according to the culture of claim 7, wherein material is selected from new LDPE (film grade), high density polyethylene(HDPE) and polypropylene, and thickness is between 2 to 8 mils.
10, according to the culture of claim 7, wherein culture is comprised in the attractive infection cell of insect, and is designed to when insect enters cell the fungi infestation insect with lethal dose.
11, according to the culture of claim 7, wherein fungi is green muscardine fungus (Metarhizium anisopliae).
12, under room temperature and comparatively high temps in packing the method for storing fungal propagulum stably over a long time, wherein packing has the oxygen level less than 5%, the relative humidity less than 10%, and wrapping material can not see through gas and water vapor.
13, according to the method for claim 12, wherein in the presence of siccative and oxygen absorbent, pack propagulum.
14,, wherein before sealing, use vacuum or pack and drain oxygen with nitrogen flush according to the method for claim 12.
15, according to the method for claim 12, wherein the fungus breeding body is that green muscardine fungus belongs to.
16, the formulation of storing fungal propagulum stably over a long time under room temperature and comparatively high temps comprises oxygen level less than 5%, and relative humidity is less than 10% packing, and wherein wrapping material can not see through gas and water vapor.
17, according to the formulation of claim 16, wherein in the presence of siccative and oxygen absorbent, pack propagulum.
18, according to the formulation of claim 16, wherein the fungus breeding body is that green muscardine fungus belongs to.
19, according to the formulation of claim 16, wherein wrapping material have the vapor transmission speed that (ⅰ) is lower than the oxygen transfer rate of 2/100 square inches/day of 0.005cc O and (ⅱ) is lower than 0.005cc water vapor/100 square inch/day.
20, under the situation that does not influence its viability, change the method for the surface properties of fungus breeding body, be characterised in that
In the conidium formulation, mix tensio-active agent, and under the condition of low relative humidity and oxygen, pack conidium.
21, according to the method for claim 20, wherein tensio-active agent is selected from hydrophilic-nonionogenic tenside, anion surfactant and the combination thereof of lipophilic thing equilibrium number in the 8-14 scope.
22, according to the method for claim 21, wherein nonionogenic tenside is selected from polymkeric substance, the polymerizing acrylic acid thing of block polymer, ethoxylated alcohol, ethoxylated alkylphenol, ethoxylated amine and acid amides, the acid of ethoxylation ester fat, ethoxylated fat family ester and oil, glyceryl ester, glycol ester, polysaccharide, polymkeric substance, propoxylation and ethoxylated fatty acid, alcohol phenol, alkylphenol, sorbitan derivatives and sucrose and the glucose ester and the derivative thereof of acrylamide.
23, according to the method for claim 21, wherein, anion surfactant is selected from the salt of sulfo-succinic acid, salt, phosphoric acid ester, tartrate and its mixture of sulfonic acid.
24, according to the method for claim 21, wherein tensio-active agent and fungus breeding body were with 1: 1000 to 100: 1 mixed.
25, according to the method for claim 21, wherein the fungus breeding body is to be obtained by the entomopathogenic fungi that is selected from muscardine and green muscardine fungus genus.
26, under the situation that does not influence its viability, change the method for the surface properties of fungus breeding body, be characterised in that
The solution that will contain tensio-active agent mixes with the fungus breeding body, is applied to the suspension of the fungus breeding body in pending zone with formation.
27, according to the method for claim 26, it further comprises suspension is applied to pending zone.
28, the method for the character on the surface of change fungus breeding body under the situation that does not influence its viability is characterised in that
The desiccated surface promoting agent is mixed, to form the dry preparation that is fit to be applied to pending zone or suspends again with the fungus breeding body.
29, according to the method for claim 28, it further comprises dry preparation is applied to pending zone.
30, form the device of fungus breeding liquid suspension, it comprises a plurality of compartments, wherein first compartment contains tensio-active agent and second compartment contains the fungus breeding body, and makes the content of the compartment mix the instrument that is applied to the fungus breeding liquid suspension in pending zone with formation mutually.
31, form the device of dry fungus breeding body preparation, it comprises a plurality of compartments, wherein first compartment contains the dry surface promoting agent and second compartment contains the fungus breeding body, and the content of the compartment is mixed mutually to form the instrument of dry fungus breeding body preparation.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/068,997 | 1993-05-17 | ||
| US08/068,997 US5989898A (en) | 1992-05-27 | 1993-05-27 | Method for storing fungal conidia |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1095758A true CN1095758A (en) | 1994-11-30 |
Family
ID=22086049
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 93116796 Pending CN1095758A (en) | 1993-05-17 | 1993-08-23 | Storing fungal cultures and conidial method |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN1095758A (en) |
| IL (1) | IL106047A0 (en) |
| ZA (1) | ZA934811B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111006991A (en) * | 2018-10-30 | 2020-04-14 | 江南大学 | Method for determining optimal storage temperature of aerobic denitrifying bacteria for sewage treatment |
| CN111165266A (en) * | 2020-01-08 | 2020-05-19 | 华南农业大学 | Method for continuously expanding propagation of arbuscular mycorrhizal fungal spores through compartment culture |
| CN112352793A (en) * | 2020-11-23 | 2021-02-12 | 重庆聚立信生物工程有限公司 | Metarhizium anisopliae granules stored for long time |
-
1993
- 1993-06-16 IL IL106047A patent/IL106047A0/en unknown
- 1993-07-05 ZA ZA934811A patent/ZA934811B/en unknown
- 1993-08-23 CN CN 93116796 patent/CN1095758A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111006991A (en) * | 2018-10-30 | 2020-04-14 | 江南大学 | Method for determining optimal storage temperature of aerobic denitrifying bacteria for sewage treatment |
| CN111006991B (en) * | 2018-10-30 | 2021-06-25 | 江南大学 | Method for determining optimal storage temperature of aerobic denitrifying bacteria for sewage treatment |
| CN111165266A (en) * | 2020-01-08 | 2020-05-19 | 华南农业大学 | Method for continuously expanding propagation of arbuscular mycorrhizal fungal spores through compartment culture |
| CN112352793A (en) * | 2020-11-23 | 2021-02-12 | 重庆聚立信生物工程有限公司 | Metarhizium anisopliae granules stored for long time |
| CN112352793B (en) * | 2020-11-23 | 2021-11-05 | 重庆聚立信生物工程有限公司 | Metarhizium anisopliae granules stored for long time |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA934811B (en) | 1994-01-25 |
| IL106047A0 (en) | 1993-10-20 |
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