CN1298224C - Fungi spore wettable dust - Google Patents
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- CN1298224C CN1298224C CNB2005100806593A CN200510080659A CN1298224C CN 1298224 C CN1298224 C CN 1298224C CN B2005100806593 A CNB2005100806593 A CN B2005100806593A CN 200510080659 A CN200510080659 A CN 200510080659A CN 1298224 C CN1298224 C CN 1298224C
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Abstract
The present invention provides the fungal spore wettable power with stable bioactivity, which is composed of insecticide fungal spore powders and auxiliary agents, wherein the wettable power comprises 50 to 75% of the insecticide fungal spore powders, and the auxiliary agents comprise a carrier, moist dispersing agents and stable protective agents, wherein the carrier is white soot, and the moist dispersing agents can be one kind or multiple kinds of substances selected from laurocapram, fatty alcohol and ethylene oxide condensation products, solid naphthalenesulfonic acid salt formaldehyde condensation products and methyl radical naphthalenesulfonic acid salt formaldehyde condensation products; additionally, the stable protective agents can be one kind or multiple kinds of substances selected from carboxymethyl cellulose, hydroxypropyl starch, methylcellulose and beta-cyclodextrin. The present invention obviously prolongs shelf lives of products, and can satisfy requirements of commercialization production.
Description
Technical field
The present invention relates to a kind of fungus insecticide, relate in particular to the wetting powder that contains the disinsection fungal spore.
Background technology
Because the long-term a large amount of of chemical pesticide use the harm to environment and mankind itself that is caused to become increasingly conspicuous, national governments have launched respectively restriction or have banned use of the policy of chemical pesticide.Biopesticide since have efficient, with environmental friendliness and advantage such as pollution-free, thereby be the more satisfactory substitute products of chemical pesticide.Very abundant in occurring in nature entomopathogen resource, it is the most important biotic factor of control insect population quantity.In the insect death that microorganism causes, disease fungus accounts for more than 60%.Therefore, the development and the exploitation microorganism insecticide particularly fungus insecticide be subjected to extensive concern both domestic and external.
Wetting powder is the most basic a kind of formulations of pesticide, can be made into suspension, sprays.It is to add a certain amount of wetting agent and inserts with the former powder of agricultural chemicals, by mechanical disruption, sieve and make.The powder of its specification requirement 99.5% is by No. 200 sieve meshes, and promptly the powder diameter should be below 7.4 * 10-5m.
Chinese patent CN02156519.8 discloses a kind of wettable powder for fungi spore.Described wetting powder by methenamine 0.3%-1.0%, lignin 5%-15%, urea 0.3%-1.0%, calcium carbonate 3.0%-10%, draw back powder 0.4%-0.8% and the fungal spore powder is formed.Described pulvis can guarantee the biologically active and the control efficiency of spore in producing, store, using.But in this patent application and storage period of not mentioned described pulvis.
Wetting powder is owing in processing, transportation, storage, plurality of advantages such as easy to use, therefore occupy significant proportion always in the formulations of pesticide.But make slow progress in disinsection fungal wetting powder research aspect.Main cause is: the active ingredient of disinsection fungal is the fungus breeding body spore or the trophosome mycelia of live body, loses vigor easily in storage process.Therefore in preparation processing, not only to improve the convenience of its use, the more important thing is how to keep the vigor of spore or mycelia, prolong the shelf life of product by preparation.In recent years, although corresponding equipment registration has abroad been arranged, the shelf life of product is still undesirable, and the disinsection fungal wetting powder of having reported both at home and abroad is no more than 12 months storage period at low temperatures.
Though China just carried out the research of beauveria bassiana wetting powder the nineties in eighties of last century,, never see its product and register also because of the problem of bin stability.Therefore, the fungus insecticide preparation of development bio-stable prolongs its bin stability and shelf life, satisfies the requirement that commercialization is produced, and is the bottleneck that promotes the industrialization of fungus insecticide product.
Summary of the invention
The problems referred to above at the prior art existence; the inventor is by the auxiliary agent of screening with the disinsection fungal biocompatible; be carrier, wetting agent, dispersant, stability protective agent and the ultra-violet absorber that uses in the preparation; optimize pharmaceutical formulation, finally developing can significant prolongation storage period and the wettable powder for fungi spore of shelf life.
Therefore, the object of the present invention is to provide a kind of wettable powder for fungi spore, its biological stability is preferable, excellent storage stability and spore content are higher.
According to one object of the present invention, the invention provides a kind of wettable powder for fungi spore of bio-stable, it is made up of disinsection fungal conidial powder and auxiliary agent, and the percentage by weight of described disinsection fungal conidial powder in wetting powder is 50～75%;
Described auxiliary agent comprises carrier, Ricinate and stability protective agent;
Described carrier is a white carbon;
Described Ricinate is to be selected from one or more of Laurocapram, fatty alcohol and ethylene oxide condensate, solid naphthalenesulfonate formaldehyde condensation compound, methyl naphthalene sulfonic acid salt formaldehyde condensation products;
Described stability protective agent is to be selected from one or more of carboxymethyl cellulose, modified starch, methylcellulose, beta-schardinger dextrin-.
Further, in whole preparation, the content of carrier is 10～15% weight, and the content of Ricinate is that the content of 5～20% weight, stability protective agent is 3～15% weight.
Described carrier can be for being selected from the biopesticide carrier than stable and inertia of this area routine, as white carbon, kaolin, diatomite and humic acid etc.
In the present invention, described carrier is preferably white carbon, the fungal spore bio-compatible that it can be used with the present invention.
But used disinsection fungal conidial powder can be the conidial powder of the fungi of any kill harmful insect among the present invention.
Described fungi can be the hyphomycetale insect pathogenic fungus usually.
Preferred, described disinsection fungal conidial powder is the conidial powder that is selected from one or more Hyphomycetes of white muscardine fungi (Beauveria), green muscardine fungus (Metarhizium), Paecilomyces varioti (Paecilomyces), Verticillium dahliae (Verticillium), wild village bacterium (Nomuraea) and quilt hair spore (Hirsutella) etc.
Most preferred, described disinsection fungal conidial powder is a beauveria bassiana spore powder.
In a specific embodiment of the present invention, used conidial powder is the beauveria bassiana conidial powder, yet it will be appreciated by those skilled in the art that, the disinsection fungal of mentioned kind has similar biological property, therefore, the prescription of wetting powder of the present invention also is applicable to the disinsection fungal spore of other kind.
The preferred Ricinate of the present invention is IW (fatty alcohol and ethylene oxide condensate) and Laurocapram, and the two ratio is 1: 1 or 1: 2, and the biocompatibility of itself and disinsection fungal spore is better.
The preferred stability protective agent of the present invention is carboxymethyl cellulose and modified starch, and both weight ratios are 1: 1～10.
Further, also can comprise ultra-violet absorber in the described wettable powder for fungi spore, for example titanium dioxide, urea, folic acid, active carbon etc., employed concentration is generally 1%.
Preferred ultra-violet absorber is a titanium dioxide.
Can also add other auxiliary agent or synergist with the fungal spore biocompatible in the preparation of the present invention, as this area thickener commonly used, antifreezing agent, bleeding agent etc.
Preferably, in the described wettable powder for fungi spore, the percentage by weight of each component is: fungal spore powder 62～75%, white carbon 10～15%, Laurocapram 5～8%, IW (fatty alcohol and ethylene oxide condensate) 3～5%, modified starch 3～5%, carboxymethyl cellulose 0.5～5%, titanium dioxide 1%.
Most preferred, in the described wettable powder for fungi spore, the percentage by weight of each component is: fungal spore powder 73.5%, white carbon 10%, fatty alcohol and ethylene oxide condensate 3.3%, Laurocapram 6.7%, carboxymethyl cellulose 0.5%, modified starch 5%, titanium dioxide 1%.
Wetting powder of the present invention can prepare with reference to the conventional preparation method of this area.In a specific embodiment of the present invention, with carrier Ricinate is adsorbed absorption in proportion fully earlier, add stability protective agent then successively, fully disperse.Finely dispersed mixture is fully mixed with the fungal spore powder again, and vacuum drying makes preparation water content≤5%.
The used disinsection fungal conidial powder of the present invention can obtain by commercially available purchase, also can cultivate or solid fermentation production disinsection fungal spore by dull and stereotyped, separates conidial powder, dry (water content≤5%), spore content 〉=1.0 * 10
11Individual/gram).
The using method of wetting powder of the present invention is that described pulvis is converted water spray, uses in the field, and concrete usage amount can be adjusted according to actual conditions, is generally 5-20 gram/mu.
The present invention screens 5 kinds of different wetting agents and dispersant Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone), IW (fatty alcohol and ethylene oxide condensate), solid NNO (naphthalenesulfonate formaldehyde condensation compound), SDS (dodecyl sodium sulfate) and MF (methyl naphthalene sulfonic acid salt formaldehyde condensation products).In a specific embodiments of the present invention, IW (fatty alcohol and ethylene oxide condensate) and Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone) all do not have obviously influence to the sprouting and the mycelial growth of beauveria bassiana spore, have good biocompatibility.
The disinsection fungal spore has metabolic activity to a certain degree and accumulates a certain amount of metabolite in storage, by selecting suitable protectant to adsorb or absorbing these to the metabolite that spore may be harmful to, spore is played a protective role.The present invention has selected 4 kinds of different stability protective agents: carboxymethyl cellulose, modified starch, methylcellulose and beta-schardinger dextrin-.In a specific embodiments, 4 kinds of stability protective agents all have in various degree facilitation to beauveria bassiana spore germination and mycelial growth; On 5.5% ratio level, modified starch and carboxymethyl cellulose have significant protective effect to the storage-stable of beauveria bassiana spore, can significantly improve the vigor of spore.
With living microorganism is that the ultraviolet ray in the daylight was the key factor that influences its vigor after the biological insecticides of main active ingredient were applied to the field.In tens hours, just lose vigor fully generally speaking.It is necessary adding a certain amount of ultraviolet-resistant absorbent in preparation.The present invention has selected 3 kinds of ultra-violet absorbers: ascorbic acid, titanium dioxide and zinc oxide.Therein in embodiment, 3 kinds of ultra-violet absorbers have in various degree facilitation to the sprouting of beauveria bassiana spore on 1% concentration level; Aspect the influence of beauveria bassiana mycelial growth, zinc oxide can promote the growth of mycelia, and titanium dioxide and ascorbic acid have certain inhibitory action; In 1～0.1% scope, titanium dioxide will significantly be better than zinc oxide and ascorbic acid to the uvioresistant protective capability of beauveria bassiana spore.
The carrier that obtains with the disinsection fungal biocompatible by above-mentioned screening is a white carbon, and Ricinate is Laurocapram and IW, and stability protective agent is modified starch and carboxymethyl cellulose, and ultraviolet-resistant absorbent is a titanium dioxide.Optimization and 15 ℃ of storage tests by pharmaceutical formulation obtain a stability and fill a prescription preferably: by weight percentage, and fungal spore powder 73.5%; White carbon (TB-2 type) 10%; IW (fatty alcohol and ethylene oxide condensate) 3.3%; Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone) 6.7%; Carboxymethyl cellulose 0.5%; Modified starch 5%; Titanium dioxide 1%.
The invention has the advantages that:
1, preparation spore content height, greater than 50%, and the spore content of the disinsection fungal wetting powder of having reported both at home and abroad is less than 50%;
2, excellent storage stability, 15 ℃ of stable down storages 18 months, the spore rate of living is still more than 70%.
In order to understand essence of the present invention better,,, describe in detail but do not limit the present invention by description to better embodiment of the present invention below in conjunction with accompanying drawing.
The embodiment of invention
The used test material of the present invention is commercially available purchase product if no special instructions.
The used fungal spore powder of the present invention can obtain by commercially available purchase, perhaps cultivates or solid fermentation by dull and stereotyped, through producing spore, collection, drying (water content≤5%, spore content 〉=1.0 * 10
11Individual/gram) back standby.Commercially available conidial powder comprises the high cryptogam of domestic fungi manufacturer (as the white muscardine fungi high cryptogam of Hubei when Yang Baijiangjunchang production), and high cryptogam standard is water content＜10%, spore content＞1,000 hundred million/gram.
The prescription screening of wettable powder for fungi spore
The vector selection of [embodiment 1] biocompatible
Select the carrier of the more stable inert carrier-white carbon (TB-2 type) of performance among the present invention as preparation.The white carbon quality is very light, has very strong absorption property, and self better water solubility is arranged, and is considered as more satisfactory biopesticide carrier both at home and abroad.In the present embodiment white carbon (TB-2 type) and beauveria bassiana biocompatibility are detected.
One, test method
(1) white carbon is to the influence of beauveria bassiana spore germination
Conidial powder and white carbon (TB-2 type) are made into spore suspension by a certain percentage, and concentration is 1.0% (w/v), and spore concentration is 1.0 * 10
6Individual/mL.Get 100 μ L spore suspensions and evenly coat on 1/4 SDAY (in w/v, glucose 1%, yeast extract 0.25%, peptone 0.25%, the agar powder 1.2%) flat board, microscopically detects the germination rate of spore behind 26 ℃ of dark 24hr of cultivation.The length of sporulation germ tube is the standard of spore germination greater than the spore diameter.The spore that adopts 100 spores countings of visual field observation at random to sprout repeats 3 times.Germination rate with pure conidial powder is contrast.With EXCEL and SPSS software data are carried out Treatment Analysis.The germination rate computational methods are as follows relatively:
Annotate: germination rate A is the germination rate of spore in certain density detected material.
(2) white carbon is to the influence of beauveria bassiana mycelial growth
White carbon (TB-2 type) added in 1/4 SDAY (in w/v, glucose 1%, yeast extract 0.25%, peptone 0.25%, the agar powder 1.2%) medium in the ratio of 1% (w/v) make flat board.It is 1.0 * 10 that Tween-80 with 0.05% is made into concentration
7The suspension of individual/mL.Get 100 μ L spore suspensions and evenly coat on the 1/4SDAY flat board, 26 ℃ of dark cultivations.Behind the 48hr, be that the card punch of 1.0cm is beaten and got lawn and be inverted together with agar block and be inoculated on the 1/4SDAY flat board that contains 1% white carbon 26 ℃ of dark cultivations with diameter.Adopt every day the right-angled intersection method to measure colony diameter, measure altogether 5 times.With the flat board that does not contain white carbon is contrast.3 repetitions are established in each processing.With EXCEL and SPSS software data are carried out Treatment Analysis.The relative growth rate computational methods are as follows:
Annotate: growth rate A is that mycelia is in the growth rate that contains on the medium of detected material.
Two, result of the test
The results are shown in Table 1.The result shows: white carbon does not all have remarkable influence to beauveria bassiana spore germination and mycelial growth under 1% concentration, has good biocompatibility with beauveria bassiana.
Table 1 white carbon carrier is to the influence of beauveria bassiana spore germination and mycelial growth
| Carrier | The relative germination rate of spore | The mycelia relative growth rate |
| Contrast | 1.00±0.00 | 1.00±0.00 |
| White carbon | 0.99±0.03 | 1.02±0.01 |
| F test | F＝16.78，P＞0.05 | F＝0.21，P＞0.05 |
Wetting agent of [embodiment 2] biocompatible and dispersant screening
In the screening process of Ricinate, except requiring that spore is had the good moistening dispersive property, because wetting agent in use directly contacts with spore with dispersant, can destroy the hydrophobic structure of spore surface, may cause certain damage to spore.So employed wetting agent and dispersant not only will have good moistening dispersiveness in the preparation, and must have good biocompatibility (not suppressing the sprouting of spore and the growth of mycelia) with spore, reduce the damage of spore as far as possible and avoid in use influencing the insecticidal effect of preparation.In this step 5 kinds of different Ricinate Laurocaprams (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone), IW (fatty alcohol and ethylene oxide condensate), solid NNO (naphthalenesulfonate formaldehyde condensation compound), SDS (dodecyl sodium sulfate) and MF (methyl naphthalene sulfonic acid salt formaldehyde condensation products) are screened.
One, biocompatibility is measured
(1) test method
1, Ricinate is to the influence of spore germination
Beauveria bassiana conidial powder and Ricinate are made into spore suspension by a certain percentage, and the concentration of each Ricinate is 1.0% (w/v), and spore concentration is 1.0 * 10
6Individual/mL.Get the 100uL spore suspension and evenly coat on 1/4SDAY (in w/v, glucose 1%, yeast extract 0.25%, peptone 0.25%, the agar powder 1.2%) flat board, microscopically detects the germination rate of spore behind 26 ℃ of dark 24hr of cultivation.The length of sporulation germ tube is the standard of spore germination greater than the spore diameter.The spore that adopts 100 spores countings of visual field observation at random to sprout.Be made as contrast with the flat board detection that does not contain Ricinate.Each is handled and repeats 3 times.With EXCEL and SPSS software data are carried out Treatment Analysis.The germination rate computational methods are as follows relatively:
Annotate: germination rate A is the germination rate of spore in certain density detected material.
2, Ricinate is to the influence of mycelial growth
To make the flat board that contains Ricinate to be detected in ratio adding 1/4SDAY (in w/v, glucose 1%, yeast extract 0.25%, peptone 0.25%, the agar powder 1.2%) medium of Ricinate in 1% (w/v).It is 1.0 * 10 that Tween-80 with 0.05% is made into concentration
7The suspension of individual/mL.Get 100 μ L spore suspensions and evenly coat on the 1/4SDAY flat board, 26 ℃ of dark cultivations.Behind the 48hr, be that the card punch of 1.0cm is beaten and got lawn and be inverted together with agar block and be inoculated on the flat board that contains Ricinate to be detected with diameter, 26 ℃ of dark cultivations adopt the right-angled intersection method to measure colony diameter every day, measure altogether 5 times.3 repetitions are established in each processing.With EXCEL and SPSS software data are carried out Treatment Analysis.The relative growth rate computational methods are as follows:
Annotate: growth rate A is that mycelia is in the growth rate that contains on the medium of detected material.
(2) result of the test
The results are shown in Table 2.As can be seen from the test results, between each Ricinate the sprouting of beauveria bassiana spore and the influence of mycelial growth all there is significant difference (F
5,12=2926.80, P＜0.05 and F
5,12=558.09, P＜0.05).SDS under 1% concentration (dodecyl sodium sulfate) and MF (methyl naphthalene sulfonic acid salt formaldehyde condensation products) have significant inhibitory effect to the sprouting and the mycelial growth of beauveria bassiana spore, and wherein SDS (dodecyl sodium sulfate) has almost completely suppressed the sprouting of spore and the growth of mycelia; IW under same concentrations (fatty alcohol and ethylene oxide condensate) and Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone) do not have significant difference with contrast, illustrate that the two and spore have relative biocompatibility preferably.
Table 2 Ricinate is to the influence of beauveria bassiana spore germination and mycelial growth
| Ricinate | Relative germination rate ± standard deviation | Relative growth rate ± standard deviation |
| Contrast | 1.00±0.00c | 1.00±0.00c |
| IW | 1.00±0.01c | 0.99±0.02c |
| SDS | 0.05±0.02a | 0a |
| MF | 0.95±0.01b | 0.57±0.05b |
| Solid NNO | 0.98±0.02bc | 0.69±0.04b |
| Laurocapram | 1.00±0.02bc | 0.96±0.02c |
| F test | F 5，12＝2926.80p＜0.05 | F 5，12＝558.09p＜0.05 |
Annotate: IW is fatty alcohol and ethylene oxide condensate; SDS is a dodecyl sodium sulfate; MF is a methyl naphthalene sulfonic acid salt formaldehyde condensation products; Solid NNO is a naphthalenesulfonate formaldehyde condensation compound; Laurocapram is 1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone; The different significance analysis of lowercase alphabet differential behind the numerical value, same row contain same letter and represent difference not remarkable (Dunnett C, p＜0.05)
Two, Ricinate is to the mensuration of the moistening dispersibility of spore
Because the surface of beauveria bassiana spore is extremely hydrophobic, should form O/W type suspension, the present invention selects in the step 2.1 2 kinds of screening to carry out different ratio optimizations with Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone), mensuration wettability and dispersiveness with the Ricinate IW (fatty alcohol and ethylene oxide condensate) of white muscardine fungi biocompatible.Proportioning between IW (fatty alcohol and ethylene oxide condensate) and the Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone) is set at 1: 1, and 1: 2,2: 1 three levels.Conidial powder, white carbon carrier and the Ricinate ratio according to 8: 1: 1 being uniformly dispersed, being testing sample, is contrast with the sample that does not add moistening dispersant.
(1) test method
The wettability assay method of Ricinate is as follows: (250mL, internal diameter add 100 ± 1mL standard water in 6.5 ± 0.5cm) to beaker.Testing sample is accurately taken by weighing 0.5g, once pour into the water, start manual time-keeping simultaneously,, write down the complete moistening time until moistening fully from the rim of a cup position.Repeat 3 times, get 3 mean value moistening time as each sample.
Dispersed assay method is as follows: testing sample is accurately taken by weighing 0.25g pour in the beaker that a certain amount of standard water is housed, after stirring with glass rod, transfer in the tool plug graduated cylinder water-bath to 30 ± 1 ℃, and add to 250mL with the standard water of same temperature, plug stopper.Graduated cylinder is put upside down (180 degree that turn upside down return original position and are changed to 1 time) 30 times in 1min.Graduated cylinder vertically is placed in the water-bath, vibration can not be arranged, no direct sunlight.Open stopper and take out 100 μ L suspension, detect the spore concentration of suspension with blood counting chamber.Behind the 30min,, measure the spore concentration in the remaining suspension with syringe sucking-off 225mL suspension in 10-15s.Repeat 3 times.The computational methods of suspensibility are as follows:
Annotate: a is the initial spore count of suspension that preparation is joined; B takes out behind the 225mL spore count of remaining preparation suspension in the graduated cylinder.
(2) result of the test
The results are shown in Table 3.Testing result shows: there were significant differences to the wettability of beauveria bassiana spore for the different proportionings of IW (fatty alcohol and ethylene oxide condensate) and Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone), wherein the quality proportioning is that moistening time of prescription of 1: 2 is the shortest, the quality proportioning is to take second place at 1: 1, and the quality proportioning is that 2: 1 used moistening time is the longest; Aspect suspensibility, 3 prescriptions do not have significant difference, all more than 90%.
Table 3 Ricinate is to the moistening of beauveria bassiana spore and dispersiveness
| Numbering | The moistening time (second) | Suspensibility (%) |
| 1 | 342±7.00b | 91.47±4.97a |
| 2 | 248±17.24a | 94.68±4.13a |
| 3 | 937±22.55c | 92.88±0.89a |
| F test | F＝1470.60，P＜0.05 | F＝0.57，P＞0.05 |
Annotate: numbering 1,2,3 refer to 1: 1 of quality proportioning between IW (fatty alcohol and ethylene oxide condensate) and the Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone) respectively, 1: 2, the different significance analysis of lowercase alphabet differential behind 2: 1 three horizontal numerical value, same row contain same letter and represent difference not remarkable (Duccan, p＜0.05)
The stability protective agent screening of [embodiment 3] biocompatible
Spore has metabolic activity to a certain degree and accumulates a certain amount of metabolite in storage, the suitable stability protective agent of adding adsorbs or absorbs these to the metabolite that spore may be harmful in preparation, and spore is played a protective role.In the present embodiment, to 4 kinds of different stability protective agents: carboxymethyl cellulose, modified starch, methylcellulose, beta-schardinger dextrin-has screened to the beauveria bassiana good biocompatibility with to the tangible macromolecular material of spore storage vigor protective effect.
One, the biocompatibility of stability protective agent and beauveria bassiana is measured
(1) test method
1, stability protective agent is to the influence of spore germination
Conidial powder and stability protective agent are made into spore suspension by a certain percentage, and the concentration of each stability protective agent is 1.0% (w/v), and spore concentration is 1.0 * 10
6Individual/mL.Get 100 μ L spore suspensions and evenly coat on 1/4SDAY (in w/v, glucose 1%, yeast extract 0.25%, peptone 0.25%, the agar powder 1.2%) flat board, 26 ℃ of dark cultivations, microscopically detects the germination rate of spore behind the 24hr.Not contain protectant spore suspension is contrast.The length of sporulation germ tube is the standard of spore germination greater than the spore diameter.The spore that adopts 100 spores countings of visual field observation at random to sprout repeats 3 times.With EXCEL and SPSS software data are carried out Treatment Analysis.The germination rate computational methods are as follows relatively:
Annotate: germination rate A is the germination rate of spore in certain density detected material.
2, stability protective agent is to the influence of mycelial growth
Stability protective agent added in 1/4 SDAY (in w/v, glucose 1%, yeast extract 0.25%, peptone 0.25%, the agar powder 1.2%) medium in the ratio of 1% (w/v) make the flat board that contains stability protective agent to be detected.It is 1.0 * 10 that Tween-80 with 0.05% is made into concentration
7The suspension of individual/mL.Get 100 μ L spore suspensions and evenly coat on the 1/4 SDAY flat board, 26 ℃ of dark cultivations.Behind the 48hr, be that the card punch of 1.0cm is beaten and got lawn and be inverted together with agar block and be inoculated on the flat board that contains stability protective agent to be detected with diameter, 26 ℃ of dark cultivations adopt the right-angled intersection method to measure colony diameter every day, measure altogether 5 times.3 repetitions are established in each processing.With EXCEL and SPSS software data are carried out Treatment Analysis.The relative growth rate computational methods are as follows:
Annotate: growth rate A is that mycelia is in the growth rate that contains on the medium of detected material.
(2) result of the test
Result of the test sees Table 4.The result shows, 4 kinds of stability protective agents that screened all have in various degree facilitation to beauveria bassiana spore germination and mycelial growth, show good biocompatibility.
Table 4 stability protective agent is to the influence of beauveria bassiana spore germination and mycelial growth
| Stability protective agent | Relative germination rate ± standard deviation | Relative growth rate ± standard deviation |
| Contrast | 1.00±0.00a | 1.00±0.00a |
| Methylcellulose | 1.01±0.05a | 1.07±0.02c |
| Carboxymethyl cellulose | 1.08±0.03b | 1.04±0.02b |
| Modified starch | 1.08±0.02b | 1.05±0.01b |
| Beta-schardinger dextrin- | 1.11±0.02b | 1.23±0.01d |
| F test | F 10，4＝8.34 p＜0.05 | F 10，4＝125.68 p＜0.05 |
Annotate: the different significance analysis of lowercase alphabet differential behind the numerical value, same row contain same letter and represent difference not remarkable (Duccan, p＜0.05)
Two, stability protective agent is to the protective effect of spore storage vigor
(1) test method
After stability protective agent and carrier white carbon and conidial powder mixed by a certain percentage, sealing was stored under 25 ℃ in the centrifuge tube of packing into, and not add the contrast that is treated to of stability protective agent, the spore vitality test is carried out in sampling after 6 months.Storage protection efficiency index computational methods are as follows:
(2) result of the test
Storage test the results are shown in Table 5.The result shows: different stability protective agent and different influences is arranged with the storage-stable to the beauveria bassiana spore between a kind of variable concentrations of stability protective agent.Wherein, be on 5.5% level in ratio, modified starch and carboxymethyl cellulose have significant protective effect to the storage-stable of beauveria bassiana spore, and are better than other 2 kinds of protectants.
Table 5 stability protective agent is to the protective effect of spore storage vigor
| Stabilizing agent | The protection efficiency index | F test | ||
| 11.1％ | 8.8％ | 5.5％ | ||
| Methylcellulose | 0.06±0.04 b | 0.01±0.03 b | 0.05±0.07 a | F＝0.07， p＞0.05 |
| Carboxymethyl cellulose | -0.35±0.08 a | 0.01±0.03 b | 0.29±0.05 b | F＝88.55， p＜0.05 |
| Modified starch | -0.13±0.05 c | -0.16±0.03 a | 0.22±0.08 b | F＝35.44， p＜0.05 |
| Beta-schardinger dextrin- | -0.15±0.06 b | -0.22±0.06 a | 0.05±0.03 a | F＝20.76， p＜0.05 |
| Ftest | F 3，8＝22.60 p＜0.05 | F 3，8＝23.15 p＜0.05 | F 3，8＝11.21 p＜0.05 |
Annotate: the different significance analysis of lowercase alphabet differential behind the numerical value, same row contain same letter and represent difference not remarkable (Duccan, p＜0.05); The protection efficiency index is that negative value represents there is not protective effect.
The screening of [embodiment 4] ultra-violet absorber
With living microorganism is that the ultraviolet ray in the daylight was the key factor that influences its vigor after the biological insecticides of main active ingredient were applied to the field, just loses vigor fully in tens hours generally speaking.Therefore, it is necessary adding a certain amount of ultraviolet-resistant absorbent in preparation.In the present embodiment, ultra-violet absorber ascorbic acid, titanium dioxide and zinc oxide are screened.
One, biocompatibility is measured
(1) test method
1, ultra-violet absorber is to the influence of beauveria bassiana spore germination
The beauveria bassiana spore is made into spore suspension with 0.05%Tween-80, then each ultra-violet absorber is added in the spore suspension respectively, and adjust concentration, make each protectant concentration be 1% (w/v), spore concentration is 1.0 * 10
7Individual/mL.Get 100 μ L spore suspensions and evenly coat on 1/4SDAY (in w/v, glucose 1%, yeast extract 0.25%, peptone 0.25%, the agar powder 1.2%) flat board, microscopically detects the germination rate of spore behind 26 ℃ of dark 24hr of cultivation.With the spore suspension that does not add ultra-violet absorber is contrast.The length of sporulation germ tube is the standard of spore germination greater than the spore diameter.The spore that adopts 100 spores countings of visual field observation at random to sprout.Repeat 3 times.With SPSS software data are carried out Treatment Analysis.The computational methods of germination rate are as follows relatively:
Annotate: germination rate A is the germination rate of spore in certain density detected material.
2, ultra-violet absorber is to the influence of beauveria bassiana mycelial growth
Ultra-violet absorber added in 1/4 SDAY (in w/v, glucose 1%, yeast extract 0.25%, peptone 0.25%, the agar powder 1.2%) medium in the ratio of 1% (w/v) make the flat board that contains ultra-violet absorber to be detected.It is 1.0 * 10 that Tween-80 with 0.05% is made into concentration
7The suspension of individual/mL.Get 100 μ L spore suspensions and evenly coat on the 1/4 SDAY flat board, 26 ℃ of dark cultivations.Behind the 48hr, be that the card punch of 1.0cm is beaten and got lawn and be inverted together with agar block and be inoculated on the flat board that contains Ricinate to be detected with diameter, 26 ℃ of dark cultivations adopt the right-angled intersection method to measure colony diameter every day, measure altogether 5 times.3 repetitions are established in each processing.With EXCEL and SPSS software data are carried out Treatment Analysis.The relative growth rate computational methods are as follows:
Annotate: A is that mycelia is in the growth rate that contains on the medium of detected material.
(2) result of the test
Biocompatibility test the results are shown in Table 6.The result shows: 3 kinds of ultra-violet absorbers have in various degree facilitation to the sprouting of beauveria bassiana spore on 1% concentration level; Aspect the influence of beauveria bassiana mycelial growth, zinc oxide can promote the growth of mycelia, and titanium dioxide and ascorbic acid have certain inhibitory action.
Table 6 ultra-violet absorber is to beauveria bassiana spore germination and the living influence of mycelia
| Ultra-violet absorber | Relative germination rate ± standard deviation | Relative growth rate ± standard deviation |
| Contrast | 1.00±0.00a | 1.00±0.00c |
| Titanium dioxide | 1.08±0.02c | 0.94±0.01b |
| Zinc oxide | 1.05±0.04bc | 1.30±0.03d |
| Ascorbic acid | 1.03±0.01ab | 0.86±0.02a |
| F test | F 8，3＝8.60，P＜0.05 | F 8，3＝308.23，P＜0.05 |
Annotate: the different significance analysis of lowercase alphabet differential behind the numerical value, same row contain same letter and represent difference not remarkable (Duccan, p＜0.05)
Two, protection potency assay
(1) test method
The beauveria bassiana spore be made into spore suspension with 0.05%Tween-80, then each ultra-violet absorber added in the spore suspension respectively, and to adjust to concentration respectively be 1%, 0.5%, 0.1% (w/v) that spore concentration is 1.0 * 10
7Individual/mL.Get 50 μ L and coat on the aseptic cover glass, 3 repetitions are established in every processing, place on the clean work station behind the natural air drying, with uviol lamp (8W, 30cm) irradiation 10min.Then postradiation cover glass is put into 10mL liquid SDY (glucose 4%, yeast extract 1%, peptone 1% are housed; W/v) shaking table is secretly cultivated (180r/min, 26 ℃) in the triangular flask of medium.Behind the 24hr, microscopically detects the germination rate of spore.Calculate the protection efficiency index, computational methods are as follows:
(2) result of the test
Result of the test sees Table 7.The result shows that titanium dioxide will significantly be better than zinc oxide and ascorbic acid to the uvioresistant protective capability of beauveria bassiana spore on 0.1～1% level.
The different ultra-violet absorbers of table 7 are to the protective effect of spore
| Ultra-violet absorber | The protection efficiency index | F test | ||
| 1％ | 0.5％ | 0.1％ | ||
| Titanium dioxide | 0.58±0.04 a | 0.45±0.08 a | 0.23±0.07 a | 22.0 (＜0.05) |
| Zinc oxide | 0.16±0.02 b | 0.13±0.05 b | 0.08±0.07 bc | 2.3 (＞0.05) |
| Ascorbic acid | 0.21±0.23 c | 0.10±0.06 b | 0.09±0.02 ab | 4.5 (＞0.05) |
| F test | F 2，2＝55.1 P＜0.05 | F 2，2＝29.6 P＜0.05 | F 2，2＝7.4 P＜0.05 | |
Annotate: the different significance analysis of lowercase alphabet differential behind the numerical value, same row contain same letter and represent difference not remarkable (Duccan, p＜0.05)
According to embodiment 1, select white carbon as carrier, the ratio in preparation is 10～15%; According to embodiment 2, select IW (fatty alcohol and ethylene oxide condensate) and wetting agent and the dispersant of Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone) as preparation, the two ratio is 1: 1 or 1: 2, and the toatl proportion in preparation is about 10%; According to embodiment 3, select carboxymethyl cellulose and modified starch stability protective agent as preparation, the ratio in preparation is about 5%; According to embodiment 4, select titanium dioxide as ultra-violet absorber, the ratio in preparation is 1%.
The preparation of wettable powder for fungi spore
The preparation of [embodiment 5] beauveria bassiana spore wetting powder
One, prescription
Constituent content (weight %)
Beauveria bassiana conidial powder 73.5%
White carbon 10%
IW 3.3％
Laurocapram 6.7%
Carboxymethyl cellulose 0.5%
Modified starch 5%
Titanium dioxide 1%
Two, preparation method
Earlier Ricinate is adsorbed absorption in proportion fully, add stability protective agent then successively, fully disperse with the carrier white carbon.Finely dispersed mixture is fully mixed with conidial powder again, and vacuum drying makes preparation water content≤5%.
Balls beauveria bassiana conidial powder can be obtained by commercially available purchase, also can be cultivated or solid fermentation by row is dull and stereotyped by beauveria bassiana, through producing spore, collection, drying (water content≤5%, spore content 〉=1.0 * 10
11Individual/gram) back standby.
The preparation of [embodiment 6] beauveria bassiana spore wetting powder
One, prescription
Constituent content (weight %)
Beauveria bassiana conidial powder 65%
White carbon 15%
IW 4.5％
Laurocapram 7.7%
Carboxymethyl cellulose 4.5%
Modified starch 3%
Titanium dioxide 1%
Two, preparation method
Referring to embodiment 5.
The preparation of [embodiment 7] beauveria bassiana spore wetting powder
One, prescription
Constituent content (weight %)
Beauveria bassiana conidial powder 70%
White carbon 12%
IW 4％
Laurocapram 6%
Carboxymethyl cellulose 3%
Modified starch 4%
Titanium dioxide 1%
Two, preparation method
Referring to embodiment 5.
The preparation of [embodiment 8] beauveria bassiana spore wetting powder
Constituent content (weight %)
Beauveria bassiana conidial powder 55%
White carbon 22%
IW 5％
Laurocapram 8%
Carboxymethyl cellulose 3%
Modified starch 5%
Titanium dioxide 2%
Two, preparation method
Referring to embodiment 5.
The preparation of [embodiment 9] Metarhizium anisopliae spore wetting powder
Constituent content (weight %)
Metarhizium anisopliae conidial powder 70%
White carbon 15%
IW 4％
Laurocapram 6%
Carboxymethyl cellulose 1%
Modified starch 3%
Titanium dioxide 1%
Two, preparation method
Earlier Ricinate is adsorbed absorption in proportion fully, add stability protective agent then successively, fully disperse with the carrier white carbon.Finely dispersed mixture is fully mixed with conidial powder again, and vacuum drying makes preparation water content≤5%.
Used Metarhizium anisopliae conidial powder can be cultivated or solid fermentation by row is dull and stereotyped by Metarhizium anisopliae, through producing spore, collection, drying (water content≤5%, spore content 〉=5.0 * 10
9Individual/gram) back standby.
The preparation of [embodiment 10] Verticillium lecanii spore wetting powder
Constituent content (weight %)
Verticillium lecanii conidial powder 65%
White carbon 15%
IW 5％
Laurocapram 8%
Carboxymethyl cellulose 1%
Modified starch 5%
Titanium dioxide 1%
Two, preparation method
Earlier Ricinate is adsorbed absorption in proportion fully, add stability protective agent then successively, fully disperse with the carrier white carbon.Finely dispersed mixture is fully mixed with conidial powder again, and vacuum drying makes preparation water content≤5%.
Used Verticillium lecanii conidial powder can be cultivated or solid fermentation by row is dull and stereotyped by Verticillium lecanii, through producing spore, collection, drying (water content≤5%, spore content 〉=2.0 * 10
9Individual/gram) back standby.
The preparation of [embodiment 11] powder Paecilomyces varioti spore wetting powder
Constituent content (weight %)
Powder Paecilomyces varioti conidial powder 68%
White carbon 12%
IW 5％
Laurocapram 8%
Carboxymethyl cellulose 2%
Modified starch 4%
Titanium dioxide 1%
Two, preparation method
Earlier Ricinate is adsorbed absorption in proportion fully, add stability protective agent then successively, fully disperse with the carrier white carbon.Finely dispersed mixture is fully mixed with conidial powder again, and vacuum drying makes preparation water content≤5%.
Used powder Paecilomyces varioti conidial powder can be cultivated or solid fermentation by row is dull and stereotyped by the powder Paecilomyces varioti, through producing spore, collection, drying (water content≤5%, spore content 〉=2.0 * 10
9Individual/gram) back standby.
[embodiment 12] Tang Pusen is by the preparation of hair spore spore wetting powder
Constituent content (weight %)
Tang Pusen is by hair spore conidial powder 75%
White carbon 11%
IW 3％
Laurocapram 6%
Carboxymethyl cellulose 1%
Modified starch 3%
Titanium dioxide 1%
Two, preparation method
Earlier Ricinate is adsorbed absorption in proportion fully, add stability protective agent then successively, fully disperse with the carrier white carbon.Finely dispersed mixture is fully mixed with conidial powder again, and vacuum drying makes preparation water content≤5%.
Used Tang Pusen can be cultivated or solid fermentation by row is dull and stereotyped by the hair spore by Tang Pusen by hair spore conidial powder, through producing spore, collection, drying (water content≤5%, spore content 〉=1.0 * 10
5Individual/gram) back standby.
The storage-stable of wettable powder for fungi spore of the present invention
[embodiment 13]
1, conidial powder preparation
Beauveria bassiana is cultivated or solid fermentation by dull and stereotyped, through producing spore, collection, drying (water content≤5%, spore content 〉=1.0 * 10
11Individual/gram) back standby.
2, preparation processing
According to embodiment 1, select white carbon (TB-2) as carrier, the ratio in preparation is 10～15%; According to embodiment 2, select IW (fatty alcohol and ethylene oxide condensate) and wetting agent and the dispersant of Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone) as preparation, the two ratio is 1: 1 or 1: 2, and the toatl proportion in preparation is about 10%; According to embodiment 3, select carboxymethyl cellulose and modified starch stability protective agent as preparation, the ratio in preparation is about 5%; According to embodiment 4, select titanium dioxide as ultra-violet absorber, the ratio in preparation is 1%.
Conidial powder and the auxiliary agent ratio in different formulations sees the following form:
Table 8 is used for the pharmaceutical formulation table of storage-stable test
| Prescription | Conidial powder | White carbon (TB-2 type) | IW | Laurocapram | Carboxymethyl cellulose | Modified starch | Titanium dioxide |
| A | 74％ | 10％ | 3.3％ | 6.7％ | 2.5％ | 2.5％ | 1％ |
| B | 73.5％ | 10％ | 3.3％ | 6.7％ | 0.5％ | 5％ | 1％ |
| C | 73％ | 10％ | 5％ | 5％ | 1％ | 5％ | 1％ |
| D | 73.5％ | 10％ | 5％ | 5％ | 0.5％ | 5％ | 1％ |
Earlier IW (fatty alcohol and ethylene oxide condensate) and Laurocapram (1-dodecyl-six hydrogen-2H-azatropylidene-2-ketone) are adsorbed absorption in proportion fully, add carboxymethyl cellulose, modified starch, titanium dioxide then successively with carrier white carbon (TB-2 type).Finely dispersed mixture is fully mixed with conidial powder again, and vacuum drying makes preparation water content≤5%.
3, the preparation storage detects with vigor
With the preparation that the Fresco Bag vacuum packaging processes, 5g/ bag, place 15 ℃, dry environment, sampling in per 2～3 months detects preparation miospore germination rate with flat band method.Through 18 months after 15 ℃, significant difference appearred in the spore vigor between 4 pharmaceutical formulations and the pure conidial powder, and the spore vigor of 4 pharmaceutical formulations all is significantly higher than contrast (pure conidial powder); Also there were significant differences between 4 pharmaceutical formulations, and the spore vigor of the C preparation of wherein filling a prescription still remains on more than 70% and (sees Table 8).
The storage test of table 8 different formulations preparation (15 ℃)
| Pharmaceutical formulation | Spore survival rate (%) | |||
| 6 months | 9 months | 14 months | 18 months | |
| Conidial powder | 96.0±1.7a | 94.3±1.5 ab | 85.0±4.6a | 6.0±2.7a |
| A | 95.7±1.5a | 97.7±1.5b | 89.0±1.0a | 58.7±3.2c |
| B | 98.7±1.5a | 93.3±1.5a | 88.0±2.9a | 72.3±4.5d |
| C | 97.7±2.5a | 96.7±2.9 ab | 88.0±3.0a | 34.3±3.2b |
| D | 98.3±2.1a | 94.0±1.0a | 84.7±4.5a | 29.3±2.1b |
| F test | F＝1.5， P＞0.05 | F＝3.2， P＞0.05 | F＝1.0， P＞0.05 | F＝193.2， P＜0.05 |
Annotate: A, B, C and D refer to the pharmaceutical formulation in the step 2.The different significance analysis of lowercase alphabet differential behind the numerical value, same row contain same letter and represent difference not remarkable (Duccan, p＜0.05).
More than the description of better embodiment of the present invention is not limited the present invention, those skilled in the art can make various changes or distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (8)
1, a kind of wettable powder for fungi spore is made up of disinsection fungal conidial powder and auxiliary agent, it is characterized in that, the percentage by weight of described disinsection fungal conidial powder in wetting powder is 50～75%;
Described disinsection fungal conidial powder is a beauveria bassiana spore powder;
Described auxiliary agent comprises carrier, Ricinate, stability protective agent and ultra-violet absorber;
Wherein said carrier is a white carbon;
Described Ricinate is to be selected from one or more of Laurocapram, fatty alcohol and ethylene oxide condensate, solid naphthalenesulfonate formaldehyde condensation compound, methyl naphthalene sulfonic acid salt formaldehyde condensation products;
Described stability protective agent is to be selected from one or more of carboxymethyl cellulose, modified starch, methylcellulose, beta-schardinger dextrin-;
Described ultra-violet absorber is to be selected from one or more of titanium dioxide, urea, folic acid, active carbon.
2, the described wettable powder for fungi spore of claim 1; it is characterized in that; in described auxiliary agent, the percentage by weight of described carrier in wetting powder is 10～15%, the percentage by weight of described Ricinate in wetting powder is 5～20%, the percentage by weight of described stability protective agent in wetting powder is 3～15%.
3, the described wettable powder for fungi spore of claim 1 is characterized in that, described Ricinate is fatty alcohol and ethylene oxide condensate and Laurocapram, and both weight ratios are 1: 1 or 1: 2.
4, the described wettable powder for fungi spore of claim 1 is characterized in that, described stability protective agent is carboxymethyl cellulose and modified starch, and both weight ratios are 1: 1～10.
5, the described wettable powder for fungi spore of claim 1 is characterized in that, the percentage by weight of described ultraviolet absorber in wetting powder is 1～2%.
6, the described wettable powder for fungi spore of claim 1 is characterized in that, described ultra-violet absorber is a titanium dioxide.
7, the described wettable powder for fungi spore of claim 6, it is characterized in that, in the described wettable powder for fungi spore, each weight percentages of components is: fungal spore powder 62～75%, white carbon 10～15%, Laurocapram 5～8%, fatty alcohol and ethylene oxide condensate 3～5%, modified starch 3～5%, carboxymethyl cellulose 0.5～5%, titanium dioxide 1%.
8, the described wettable powder for fungi spore of claim 7, it is characterized in that, in the described wettable powder for fungi spore, each weight percentages of components is: fungal spore powder 73.5%, white carbon 10%, Laurocapram 6.7%, fatty alcohol and ethylene oxide condensate 3.3%, modified starch 5%, carboxymethyl cellulose 0.5%, titanium dioxide 1%.
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| CN104738097A (en) * | 2015-03-07 | 2015-07-01 | 吉林省农业科学院 | Beauveria bassiana barrel mixed agent and preparation method thereof |
| CN110214793A (en) * | 2019-04-26 | 2019-09-10 | 云南微态源生物科技有限公司 | Thick wall cell verticil branch bacterium biological pesticide novel form and preparation method thereof |
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| CN101564047B (en) * | 2009-05-27 | 2012-02-15 | 重庆大学 | Disinsection fungi emulsifiable powder |
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| CN1415210A (en) * | 2002-12-16 | 2003-05-07 | 张泽华 | Wettability powder of fungi spore |
| CN1491539A (en) * | 2002-10-25 | 2004-04-28 | 组合化学工业株式会社 | Agricultural wettable composition, its producing method and preserving method |
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| CN1415210A (en) * | 2002-12-16 | 2003-05-07 | 张泽华 | Wettability powder of fungi spore |
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| CN104738097A (en) * | 2015-03-07 | 2015-07-01 | 吉林省农业科学院 | Beauveria bassiana barrel mixed agent and preparation method thereof |
| CN110214793A (en) * | 2019-04-26 | 2019-09-10 | 云南微态源生物科技有限公司 | Thick wall cell verticil branch bacterium biological pesticide novel form and preparation method thereof |
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