CN100341413C - Beauveria bassiana preparation used for preventing and controlling pine moth, its preparation method and use - Google Patents
Beauveria bassiana preparation used for preventing and controlling pine moth, its preparation method and use Download PDFInfo
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Abstract
The present ivnention relates to a bassiana preparation for controlling pine moths, particularly to a nutritional type wet bacterial powder bassiana preparation for controlling pine moths, which is suitable for north arid area. The preparation uses the strain of beauveria bassiana of which the preservation number is CGMCC NO. 1573 and which is formed by solid fermentation and preparation. The production process of the present invention comprises that the nutritional type wet bacterial powder is prepared by mixing the nutritional type wet filling materials with the strain which is treated by primary slant culture, secondary canning culture and tertiary bagging culture, products are used for controlling pine moths in the forest, and the bacterial powder is uniformly broadcast below the tree base litter for pine moth hibernation; the consumption is 150 to 250 g/strain, and the control efficiency reaches 70 to 85 percent. The preparation is suitable for characteristics of north forest environment, provides nutrition and water required by living and breeding strain on the forest land, and improves the opportunities of strain vitality and successful infection so as to have the advantages of less strain consumption, simple method, no pollution, etc.
Description
Technical field
The present invention relates to a kind of microorganism formulation, specifically belong to a kind of white muscardine fungi preparation that is used to prevent and treat pine caterpillars, particularly belong to a kind of white muscardine fungi preparation that is applicable to dry area in north china control pine caterpillars, and its preparation method and application.
Background technology
Pine forest is the main type of China forest, is the main contents of wildwood protected project, and the ecological safety of China is played an important role, and especially in northern ecologically fragile areas, the barrier action of pine forest performance is huge.But pine caterpillars is the primary pest of pine forest always.China pine caterpillars has 27 kinds (containing 4 subspecies), and it is the widest to distribute with dendrolimus punctatus in south, endangers the heaviest.The north is that dendrolimus tabulaeformis, dendrolimus spectabilis, Huashan pine caterpillar and dendrolimus sibiricus harm are the heaviest, causes disaster year after year, is called as " smokeless forest fire ".Many in the past employing chemical insecticide control pine caterpillars cause environmental pollution, residue of pesticide, killed natural enemies, make insect develop immunity to drugs, form again a series of problems such as wildness.Using insect pathogenic bacteria and carry out biological control, is the important channel of realizing forest health and insect sustainable Control.In the world this is attached great importance to the special U.S., Japan, Europe etc. in prosperity.China has just carried out the research that utilizes white muscardine fungi control pine caterpillars since the seventies in 20th century, has obtained very great achievement.But,, it is used still is confined to 12 provinces and regions, south so far, based on Control Dendrolimus punctatus because the white muscardine fungi growth and breeding needs higher humidity and suitable temperature.The main cause that north restriction white muscardine fungi is used has 2, and the first, lack the local excellent species that adapts to blreal forest arid low temperature environment.The white muscardine fungi bacterial classification of using in south is to separate from the dendrolimus punctatus that catches an illness to obtain, and they generally have the southern warm of adaptation and adapt to genetic characteristics parasitic on former host dendrolimus punctatus, and the north can not finely be adapted to.The second, lack the dosage forms and the application technique that are fit to the blreal forest environmental applications.At present, in the hot and humid environment of southern forest, spray spore suspension, the spore finish of white muscardine fungi or spray the conidial powder of doing, spore can be survived and realization is infected, and reaches control efficiency.But in the north because the dry low temperature in the forest, use above-mentioned formulation and method after, the spore of white muscardine fungi can be very fast thirst or low temperature is freezed to death because of dehydration, or because of absorbing less than moisture, spore can not be sprouted or vigor reduces, and can not successfully infect, and makes the control failure.Therefore, just must on above-mentioned two limiting factors, realize breaking through northern successful Application white muscardine fungi control pine caterpillars, naturally the bacterial classification of the pine caterpillars cause of disease white muscardine fungi that survives in the promptly necessary acquisition blreal forest environment is developed the special preparation and the application corresponding technology that adapt to the blreal forest characteristics.
Summary of the invention
The object of the present invention is to provide a kind of white muscardine fungi preparation of preventing and treating pine caterpillars, particularly be applicable to white muscardine fungi preparation of dry area in north china control pine caterpillars and its production and application.
A kind of white muscardine fungi preparation of preventing and treating pine caterpillars provided by the invention, adopting preserving number is beauveria bassiana (Beauveria bassiana) bacterial classification of CGMCC No.1573, by solid culture, that culture and the wet filler of nutrition type is formulated.
Described bacterial classification is that the inventor adopts the corpse separation acquisition that the dendrolimus tabulaeformis of surviving the winter is caught an illness naturally from March, 2002 in the artificial Chinese pine woods in forest farm, Lingqiu County, northern territory, Shanxi Province, is accredited as beauveria bassiana (Beauveriabassiana).The bacterial strain of this bacterial classification has been deposited in Beijing China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on December 22nd, 2005, culture presevation number is CGMCC No.1573.
The preparation method of white muscardine fungi preparation of the present invention comprises the steps:
(1) primary inclined plane spawn culture: with preserving number is that the beauveria bassiana bacterial classification of CGMCC No.1573 inserts and to be equipped with in the test tube of the PDA slant medium that contains healthy oil pine caterpillars worm corpse leachate and peptone, put into incubator, temperature is that 20 ℃-28 ℃, relative moisture are to cultivate under the 75%-85% condition, treats when mycelia is covered with the inclined-plane and produces spore standby; General incubation time is 10-15 days.
(2) secondary solid culture: under aseptic condition, cultured slant strains and medium thereof together inserted in the Cans that solid culture medium is housed cultivate, per 1 Cans inoculum concentration is 1 test tube slant bacterial classification, it is that 20 ℃-28 ℃, relative moisture are to be cultured under the 75%-85% condition to cover with white hypha that postvaccinal Cans are placed temperature, makes second class inoculum; General incubation time is 20-23 days.
(3) three grades of solid enlarged culture: with second class inoculum, be 1 by volume: the inoculum concentration of 10-25 inserts in the solid culture medium of the bacterium of going out, in temperature is that 20 ℃-28 ℃, relative moisture are to be cultured under the 75%-85% condition to cover with white hypha, promptly gets three grades of cultures; General incubation time is 25-30 days.
(4) the wet bacterium powder preparation of nutrition type: with raw materials by weight meter wheat bran 68-72, husk 26-30, milk powder 0.5-1.5, water-loss reducer 0.5-1.5 mixing, and add water 60%-80% by the raw material gross weight and stir, sterilization, be cooled to room temperature, make the wet filler of nutrition type; Again three grades of cultures and nutrition type are wet filler in 1: the 4-6 ratio stirs in desinfection chamber, and packing promptly gets the stiff bacteria preparation product of the wet bacterium whitewash of nutrition type;
Wherein, the solid culture medium described in step (2), (3) is: with raw materials by weight meter wheat bran 60-80, and husk 20-40 mixing, and add water 60%-80% by the raw material gross weight and stir, sterilization makes.
Described slant culture based component and proportion optimization thereof are: potato 200g, glucose 20g, agar 20g, peptone 20g, the dried worm corpse of pine caterpillars larva 30g, water 1000mL.
Described secondary and three grades of solid culture medium material compositions and proportioning thereof are preferably by weight percentage: wheat bran 70, husk 30.
Wet filler material composition of described nutrition type and proportioning thereof are preferably by weight percentage: wheat bran 70, husk 28, milk powder 1, water-loss reducer 1.Wherein, water-loss reducer is selected from cross-linked polyacrylamide, polyacrylate (sylvite or sodium salt) and polyacrylamide EVA, " section is vast by 98 " efficient drought-resistant water-loss reducer etc.
The stiff bacteria preparation of the wet bacterium whitewash of nutrition type of the present invention can be used for preventing and treating pine caterpillars, particularly is applicable in the dry area in north china control pine caterpillars and promotes the use of.The present invention also can be used for using in the southern climates humid region control pine caterpillars certainly.
The woodland application process of white muscardine fungi preparation: take the stiff bacteria preparation of the wet bacterium whitewash of the nutrition type for preparing to woodland that pine caterpillars is taken place, the litter that tree root base portion pine caterpillars is hidden digs out gently, evenly spreads fertilizer over the fields the bacterium powder, and then litter built gets final product.Consumption is the 150-250g/ strain, decides according to the size of tree dish.Application time since winter, November, pine caterpillars survived the winter, until by the end of March can next spring.
Effect is checked: mid-April when pine caterpillars comes out of hibernation climb the tree before, adopt the methods of sampling to check control efficiency.The healthy worm and the dead quantity percentage of catching an illness of living of counting calculates corrected mortality according to contrast again.General preventive effect is between 65%-85%.In addition, also dead at that time pine caterpillars also can continue death owing to carry disease germs after coming out of hibernation, and forms the further expansion propagation of bacterial classification.Bacterial classification can continue to play a role at woodland after use, reaches the long-term effect of biological Sustainable Control.
Advantage of the present invention and effect assessment:
1, the stiff bacteria preparation of the wet bacterium whitewash of applied nutrition type has very good effect spring and control in the winter pine caterpillars larva that survives the winter, and makes the larva lethality of catching an illness reach 65%-85%.Illustrate that the wet bacterium powder preparation of applied nutrition type is the effective ways of northern area control pine caterpillars biological control, it has broken through the restriction of blreal forest environmental drying low temperature to the application of disease fungus.
2, the stiff bacteria preparation of the wet bacterium whitewash of the nutrition type nutrition and the moisture that directly provide white muscardine fungi in the forest land, to need most, use the back to improving the subenvironment of white muscardine fungi existence under the litter at woodland, to survival, the growth of preserving white muscardine fungi, breed and infect and have important function.The quantity of white muscardine fungi is increased a lot, form dominant bacteria group, the chance of bacteria infection is improved greatly.
3, the stiff bacteria preparation of the wet bacterium whitewash of applied nutrition type can not only cause direct control to contemporary dendrolimus tabulaeformis larva in the forest land, and it can be in long preservation under the litter, the larva that survives the winter the coming year formed once more infect, also can be but the pine caterpillars larva that can also continue to come out of hibernation is carried to tree crown or other positions by microbiological contamination, form to propagate.Therefore, by manually spreading fertilizer over the fields the wet bacterium powder preparation of nutrition type, can increase the bacterium amount of woodland greatly, rely on the mutual infection between this gregariousness and the polypide, pathogen transmission faster impels epidemic disease to take place, and forms the good effect of Sustainable Control.
4, the stiff bacteria preparation of the wet bacterium whitewash of applied nutrition type is prevented and treated for the dendrolimus tabulaeformis of surviving the winter under the tree root, and it is concentrated relatively also to have a bacteria using amount of executing, and scope is little, operate easily and the saving of labor, biological control is pollution-free, and whole winter and early spring all can be used, and has utilized advantages such as rural area and forest zone surplus labour force.
In a word, production technology of the present invention is simple, operation easily, and raw material is obtained easily, and is with low cost, pollution-free, and products obtained therefrom control pine caterpillars effect is remarkable, particularly is applicable in the dry area in north china control pine caterpillars to use in a large number, and popularizing application prospect is wide.
The bacterial classification explanation
The bacterial strain of the beauveria bassiana (Beauveria bassiana) that the present invention adopts has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on December 22nd, 2005, culture presevation number is CGMCCNo.1573.The preservation centre address: in No. 13 institutes of Institute of Microorganism, Academia Sinica in north, BeiJing ZhongGuanCun, postcode: 100080.
Embodiment
The preparation of the stiff bacteria preparation of the wet bacterium whitewash of embodiment 1 nutrition type of the present invention
(1) primary inclined plane spawn culture:
1, the preparation of slant medium:
Slant culture based component and proportioning thereof: potato 200g, glucose 20g, agar 20g, peptone 20g, dried worm corpse 30g, water 1000mL.Its preparation process:
1., claim 3-5 the dried worm corpse of the dendrolimus tabulaeformis larva 30g in age, adding 1000mL clear water immersion 2 hours.
2., take by weighing 200g after peeling potatoes dug bud, be cut in the dried worm corpse liquid that joins immersion behind the fritter of the about 1cm of length and width and boil altogether, use filtered through gauze, get filtrate to the potato back of feeling like jelly.
3., with this filtrate supplementing water to 1000mL, add the 20g peptone, heating is until whole dissolvings.
4., add agar 20g, adding 20g glucose after dissolving fully, filtered through gauze in the boiling part.Medium is the nature pH value.
5., filtrate passes through to account for 1/5 of test tube in the funnel injecting tube.
6., test tube 1.5Kg/cm
2Autoclaving 30min.Taking-up is put into inclined-plane (highly be about test tube 1/2) with test tube.
7., treat the medium cooled and solidified after, put into 25 ℃ of constant incubators and cultivate 72h, do aseptic investigation.Get qualified test tube then and prepare inoculation.
2, primary inclined plane spawn culture: with preserving number is that the beauveria bassiana of CGMCC No.1573 is inoculated in the test tube that above-mentioned slant medium is housed, put into incubator, in temperature is that 25 ℃, relative moisture are under 75% condition, cultivated 12 days, when treating that mycelia is covered with the inclined-plane and produces spore, change the solid secondary over to and cultivate.
(2) the canned cultivation of secondary solid:
1, the preparation of solid culture medium in the secondary solid culture:
Secondary solid culture medium material composition and proportioning thereof: wheat bran 7Kg, husk 3Kg.Its preparation process:
1., two kinds of raw materials weigh up water mixing in proportion and mix, amount of water is equivalent to the 60%-80% of raw material gross weight, up to hold just dripped when grabbing till, stir and be loaded on Cans after even.
2., the Cans middle body disclose a glance to the bottle end, be convenient to colony growth, bottleneck is built with four layers of gauze and two-layer brown paper and is tightened.
3., use 1.5Kg/cm
2Autoclaving 40 minutes, take out the sterilization back, is cooled to room temperature, can inoculate under gnotobasis.
2, the canned cultivation of secondary solid: bacterial strain in the cultured test tube is connected medium together scrape and put into the Cans that above-mentioned secondary solid culture medium is housed and cultivate under aseptic condition, inoculum concentration is one bottle of every test tube strains inoculation, postvaccinal Cans are put into constant incubator, in temperature is that 25 ℃, relative moisture are under 75% condition, cultivated 20 days, and in culture tank, covered with white hypha.
(3) three grades of packed enlarged culture of solid:
1, the preparation of solid culture medium in three grades of solid enlarged culture:
Three grades of solid culture medium material compositions and proportioning thereof: wheat bran 35Kg, husk 15Kg.Its preparation process:
1., raw material weighs up water mixing in proportion and mixes, amount of water is equivalent to the 60%-80% of raw material gross weight, up to hold just dripped when grabbing till, stir and be loaded in three grades of culture bag after even.The material of culture bag is poly-third ethene, and diameter is 20cm, the long 30cm of bag.The sack sealing.
2., use 1.5Kg/cm
2Autoclaving 40min, take out sterilization back, is cooled to room temperature, can inoculate under gnotobasis.
2, three grades of packed enlarged culture of solid: with cultured second class inoculum, connect 7 bags (being equivalent to inoculum concentration volume ratio 1: 20) by every bottle of bacterial classification, insert in three grades of culture bag of the above-mentioned bacterium of going out, be in temperature that 25 ℃, relative moisture are 75%, under the aseptic condition, cultivated 25 days, and treated that mycelia and spore covered with culture bag and promptly get three grades of cultures.
(4) preparation of the wet bacterium powder of nutrition type:
1, the preparation of the wet filler of nutrition type:
Wet filler material composition of nutrition type and proportion optimization are: wheat bran 70%, husk 28%, milk powder 1%, water-loss reducer cross-linked polyacrylamide 1%.Its preparation process:
1., raw material is mixed in proportion, and add water 60%-80% by the raw material gross weight and stir.
2., use 1.5Kg/cm
2Autoclaving 40min, sterilization is cooled to room temperature, makes the wet filler of nutrition type.
2, the preparation of the wet bacterium powder of nutrition type: with three grades of cultures, in culture bag, take out, in sealed stirrer, mixes by 1: 5 ratio and stir, promptly obtain the nutrition type bacterium whitewash deadlock bacteria preparation that wets with the wet filler of nutrition type.Be distributed into bag then, can arrive woodland and use.The material of bag is a polyvinyl chloride, diameter 25cm, long 40cm, the every packed nutrition type bacterium powder 2.5kg that wets.
The woodland of the stiff bacteria preparation of the wet bacterium whitewash of embodiment 2 nutrition types is used test:
(1) use the northern territory in winter of the stiff bacteria preparation of the wet bacterium whitewash of nutrition type: use in the 35 years stranger worker Chinese pines woods of Western Hills mineral office forest farm, Lingqiu County, northern territory, Shanxi Province in November, 2003, the control area is 15 hectares.The wet bacterium powder of nutrition type for preparing being taken to the woodland of pine caterpillars generation uses.The litter that tree root base portion pine caterpillars is hidden digs out gently, evenly spreads fertilizer over the fields the bacterium powder, and then litter built gets final product.Consumption is the 250g/ strain.
Effect is checked: late April when pine caterpillars comes out of hibernation climb the tree before, adopt the methods of sampling to check control efficiency.The counting health worm alive and the dead quantity percentage of catching an illness calculate according to contrast and proofread and correct death, and preventive effect is at 70.4%-85.4%.
(2) use the northern territory in spring of the stiff bacteria preparation of the wet bacterium whitewash of nutrition type:
In late March, 2004 is used in the 35 years stranger worker Chinese pines woods of Western Hills mineral office forest farm, Lingqiu County, northern territory, Shanxi Province, and the control area is 10 hectares.The wet bacterium powder of nutrition type for preparing being taken to the woodland of pine caterpillars generation uses.The litter that tree root base portion pine caterpillars is hidden digs out gently, evenly spreads fertilizer over the fields the bacterium powder, and then litter built gets final product.Consumption is the 150g/ strain.
Effect is checked: late April when pine caterpillars comes out of hibernation climb the tree before, adopt the methods of sampling to check control efficiency.The counting health worm alive and the dead quantity percentage of catching an illness calculate according to contrast and proofread and correct death, and preventive effect is at 47.1%-54.5%.
(3) use the southern areas in spring of the stiff bacteria preparation of the wet bacterium whitewash of nutrition type:
In mid-March, 2005, in Duan Shi forest farm 40 years stranger worker Chinese pines woods of Shanxi Province region of Southeast Zhongtiao Shan Mountain forest-management office administration, to use, the control area is 10 hectares.The wet bacterium powder of nutrition type for preparing being taken to the woodland of pine caterpillars generation uses.The litter that tree root base portion pine caterpillars is hidden digs out gently, evenly spreads fertilizer over the fields the bacterium powder, and then litter built gets final product.Consumption is the 150g/ strain.
Effect is checked: late April when pine caterpillars comes out of hibernation climb the tree before, adopt the methods of sampling to check control efficiency.The counting health worm alive and the dead quantity percentage of catching an illness calculate according to contrast and proofread and correct death, and preventive effect is at 62.5%-65.9%.
In addition, also dead at that time pine caterpillars also can continue death owing to carry disease germs after coming out of hibernation, and forms the further expansion propagation of bacterial classification.Bacterial classification can continue to play a role at woodland after use, reaches the long-term effect of biological Sustainable Control.
Claims (5)
1, a kind of white muscardine fungi preparation that is used to prevent and treat pine caterpillars is characterized in that, adopting preserving number is beauveria bassiana (Beauveria bassiana) bacterial classification of CGMCC No.1573, is prepared from by the following method:
(1) primary inclined plane spawn culture: with preserving number is that the beauveria bassiana bacterial classification of CGMCC No.1573 inserts and to be equipped with in the test tube of the PDA slant medium that contains healthy oil pine caterpillars worm corpse leachate and peptone, put into incubator, temperature is that 20 ℃-28 ℃, relative moisture are to cultivate under the 75%-85% condition, treats when mycelia is covered with the inclined-plane and produces spore standby;
(2) secondary solid culture: under aseptic condition, cultured slant strains and medium thereof together inserted in the Cans that solid culture medium is housed cultivate, per 1 Cans inoculum concentration is 1 test tube slant bacterial classification, it is that 20 ℃-28 ℃, relative moisture are to be cultured under the 75%-85% condition to cover with white hypha that postvaccinal Cans are placed temperature, makes second class inoculum;
The inoculum concentration of (3) three grades of solid enlarged culture: with second class inoculum, be 1 by volume: 10-25 inserts in the solid culture medium, is to be cultured under the 75%-85% condition to cover with white hypha at 20 ℃-28 ℃, relative moisture, promptly gets three grades of cultures;
(4) the wet bacterium powder preparation of nutrition type: with raw materials by weight meter wheat bran 68-72, husk 26-30, milk powder 0.5-1.5, cross-linked polyacrylamide 0.5-1.5 mixing, and add water 60%-80% by the raw material gross weight and stir, sterilization, be cooled to room temperature, make the wet filler of nutrition type; Again three grades of cultures and nutrition type are wet filler in 1: the 4-6 ratio stirs in desinfection chamber, and packing promptly gets the stiff bacteria preparation product of the wet bacterium whitewash of nutrition type;
Wherein, the solid culture medium described in step (2), (3) is: with raw materials by weight meter wheat bran 60-80, and husk 20-40 mixing, and add water 60%-80% by the raw material gross weight and stir, sterilization makes;
Slant culture based component and proportioning thereof described in the step (1): potato 200g, glucose 20g, agar 20g, peptone 20g, the dried worm corpse of pine caterpillars 30g, water 1000mL.
2, according to the described white muscardine fungi preparation of claim 1, it is characterized in that, described solid culture medium material composition and proportioning by weight percentage: wheat bran 70, husk 30.
3, according to the described white muscardine fungi preparation of claim 1, it is characterized in that, wet filler material composition of described nutrition type and proportioning by weight percentage: wheat bran 70, husk 28, milk powder 1, cross-linked polyacrylamide 1.
4,, be used to prevent and treat pine caterpillars according to the purposes of the described white muscardine fungi preparation of claim 1.
5, the purposes of white muscardine fungi preparation as claimed in claim 4 is used for northern area forest land control pine caterpillars.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102634459B (en) * | 2012-02-29 | 2013-09-18 | 天津市植物保护研究所 | Beauveria bassiana strain having high virulence to fall webworm and application thereof |
| CN102690760A (en) * | 2012-06-06 | 2012-09-26 | 山西大学 | Beauveria bassiana preparation for controlling peach fruit borers and preparation method thereof |
| CN106508997A (en) * | 2016-10-31 | 2017-03-22 | 郑州思辩科技有限公司 | Microorganism wettable powder for preventing pine caterpillars and preparation method thereof |
| CN107347922A (en) * | 2017-08-18 | 2017-11-17 | 临沂市农业科学院 | A kind of application of Strain of Beauveria bassiana |
| CN109810907B (en) * | 2019-03-18 | 2020-07-31 | 福建农林大学 | Beauveria bassiana BbJ L-01 with strong pathogenicity on terminal-age larvae of cryptomeria fortunei caterpillars |
| CN112920956B (en) * | 2021-04-28 | 2022-08-19 | 河北农业大学 | Beauveria bassiana BD01 strain and fermentation method and application thereof |
| CN114456814A (en) * | 2022-01-17 | 2022-05-10 | 聊城金余肥业有限公司 | Low-density water-retention insect-resistant saline-alkali soil conditioner and preparation method thereof |
| CN114507096A (en) * | 2022-01-17 | 2022-05-17 | 聊城金余肥业有限公司 | Low-density vegetable organic bacterial fertilizer and preparation method thereof |
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| CN1395839A (en) * | 2002-05-13 | 2003-02-12 | 中国农业科学院生物防治研究所 | Preparing process and usage of muscardine insecticide |
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