CN102690760A - Beauveria bassiana preparation for controlling peach fruit borers and preparation method thereof - Google Patents
Beauveria bassiana preparation for controlling peach fruit borers and preparation method thereof Download PDFInfo
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Abstract
The invention provides a beauveria bassiana preparation for controlling peach fruit borers and a preparation method thereof. The preparation method comprises the following steps of: inoculating a strain of beauveria bassiana with the collection number of CGMCC No. 4526 into a test tube loaded with a PDA (potato dextrose agar) slant culture medium containing a healthy peach fruit borer corpse leachate for culture, further performing second-stage strain culture and third-stage solid amplification culture, and finally preparing a bacterial powder preparation. The bacterial powder preparation provided by the invention has a better effect on controlling the peach fruit borers, the control effect can be up to 90.5%, and the fruit-boring rate of an orchard in the same year can be reduced by 83.8% -93.6%. The bacterial powder preparation provided by the invention is particularly suitable for controlling the peach fruit borers in dry and low-temperature regions in the north.
Description
Technical field
The present invention relates to microbial preparation, specifically belong to a kind of beauveria bassiana preparation that is used to prevent and treat small heart-eating peach worm and preparation method thereof.
Background technology
Small heart-eating peach worm (it is little to be called for short peach) is the serious fruit tree budworm class pest of harm.Generally take place in northern orchard, the fruit of more than 10 kind of fruit trees such as harm apple, jujube, pears, peach, apricot, hawthorn causes very big financial loss to the orchard worker.Mainly used chemical insecticide that peach is prevented and treated for a short time in the past.Not only can cause environmental pollution, killed natural enemies insect, make a series of problems such as insect develops immunity to drugs.But also can cause pesticide residue, reduce the quality of fruit.Using insect pathogenic bacteria and carry out biological control, is the important channel of realizing the insect sustainable Control.China has just begun to utilize the research of muscardine control small heart-eating peach worm since the seventies in 20th century, has obtained very great achievement.
But first because small heart-eating peach worm is a moth fruit insect, and the whole larval growth phase is all inner at fruit.And the method for preventing and treating now commonly used is to detect unearthed Sheng phase of the larva that survives the winter and adult after the apparition with the monitoring and prediction method earlier, uses chemistry or biotic pesticide again, need spray in a season 2 ~ 3 times.Need bigger man power and material to monitor and implement.The second, because the muscardine growth and breeding generally needs higher humidity and suitable temperature.Just require to adapt to the local excellent species of northern arid low temperature environment northern successful Application muscardine control small heart-eating peach worm; Naturally the bacterial classification of the small heart-eating peach worm cause of disease muscardine that survives in the northern environment of promptly necessary acquisition is developed the utilisation technology that adapts to northern area.
Summary of the invention
The object of the present invention is to provide a kind of muscardine preparation of preventing and treating small heart-eating peach worm, particularly be applicable to the muscardine preparation of northern arid low temp area control small heart-eating peach worm, and the method for preparing said preparation.
A kind of muscardine preparation of preventing and treating small heart-eating peach worm provided by the invention is to adopt preserving number to be beauveria bassiana (Beauveria bassiana) bacterial classification of CGMCC No.4526, and is through solid culture, that culture and nutrient filler is formulated.
Described bacterial classification; Being contriver 2009 separates and obtains from picking up from the peach of falling ill naturally in apple orchard, Xiangfen County, the Shanxi Province larva that survives the winter for a short time; Classification name: beauveria bassiana (Beauveria bassiana); The bacterial strain of this bacterial classification has been deposited in No. 3 China Committee for Culture Collection of Microorganisms in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City common micro-organisms center (CGMCC) on January 5th, 2011, culture presevation number is CGMCC No.4526.
A kind of preparation method who prevents and treats the muscardine preparation of small heart-eating peach worm provided by the invention comprises the steps:
(1) primary inclined plane spawn culture: with preserving number is that the beauveria bassiana bacterial classification of CGMCC No.4526 inserts and to be equipped with in the test tube of the PDA slant medium that contains healthy small heart-eating peach worm worm corpse leach liquor; Put into incubator; In temperature is that 20 ℃-28 ℃, relative humidity are to cultivate under the 70%-85% condition, treats when mycelia is covered with the inclined-plane and produces full spore subsequent use; General incubation time is 8-12 days.
(2) second class inoculum is cultivated: under aseptic condition; Cultured primary inclined plane bacterial classification is scraped together with substratum; Put into the triangular flask that contains liquid nutrient medium and cultivate, inoculum size is put into the isothermal vibration incubator for 3 bottles of each test tube strains inoculations with postvaccinal triangular flask; 20 ℃-28 ℃, 150-200r/min are cultured to when covering with mycelium pellet in the nutrient solution subsequent use; General incubation time is 10-15 days.
(3) three grades of solid enlarged culturing: under aseptic condition; With second class inoculum; Be 1 by volume: the inoculum size of 10-25 inserts in the solid medium of the bacterium of going out; In temperature is that 20 ℃-28 ℃, relative humidity are to cultivate under the 70%-85% condition, to substratum, covers with white hypha, promptly gets three grades of cultures; General incubation time is 25-30 days.
(4) bacterium powder preparation preparation: with raw materials by weight meter wheat bran 60-80, husk 20-40 (preferred: wheat bran 70, husk 30) mixing, and add water 60%-80% by the raw material gross weight and stir, sterilization, be cooled to room temperature after, promptly get filler; With filler and three grades of cultures in 1-3: 1 ratio stirs in sterilisable chamber, and packing promptly gets muscardine bacterium powder preparation;
Wherein, the described PDA slant medium proportioning that contains healthy small heart-eating peach worm worm corpse leach liquor is: yam 200g/L, glucose 20g/L, agar 20g/L, the dried worm corpse of small heart-eating peach worm larva 10g/L.
Described liquid nutrient medium is the SDAY substratum, and its composition and proportioning are: peptone 10g/L, yeast extract paste 2g/L, glucose 40g/L;
Described solid medium makes through following method: by weight percentage, and with raw material wheat bran 68-72, husk 26-30; Milk powder 1-4 (preferred: wheat bran 70, husk 28, milk powder 2) mixing; And the water of adding raw material gross weight 60%-80%, stirring, sterilization makes.
Muscardine bacterium powder preparation of the present invention can be used for preventing and treating small heart-eating peach worm, particularly is applicable to northern arid low temp area control small heart-eating peach worm, also can be used for weather warm and moist area control small heart-eating peach worm and uses.Control effect can reach 82.1%-90.5%.The orchard borer fruit rate reduces 83.8%-93.6% then.
The woodland application process of muscardine preparation: the thick veneer of soil of 1-2cm peripheral about 1 meter of the tree root base portion that small heart-eating peach worm is taken place is pushed aside; Evenly spread fertilizer over the fields after muscardine bacterium powder preparation and soil mixed thoroughly in 1 ︰ 0.5-1.5 ratio, and then veneer of soil built get final product; Consumption is the 150-250g/ strain, time of application be autumn 8, September small heart-eating peach worm will take off fruit bury survive the winter before.Can survive the winter spring 3, April small heart-eating peach worm imposes once before larva is unearthed, and improves control effect, and consumption is the 100-200g/ strain.
We are chosen in autumn 8, September small heart-eating peach worm larva and from fruit, come out to get into and the muscardine bacterium powder preparation for preparing is manured into soil before surviving the winter in the soil, can make like this infection chance be increased to larva bury knot survive the winter before the cocoon and take off cocoon after be unearthed twice.Because muscardine has stronger place to deposit ability and more stable virulence and product spore level in soil.8, be manured into soil September in the virulence of beauveria bassiana spore can last till and continue preinfection June in the coming year not by the peach small grub after infecting, surviving the winter.In addition because the infection lethal effect of muscardine needs the regular hour, and the life cycle of small heart-eating peach worm larva in soil is very long, and the phase is infected lethal effect to the larva performance of surviving the winter at this moment can to let muscardine.And after the infected larva death, muscardine can also grow and produce spore from the worm corpse, increases beauveria bassiana spore content in the soil, makes it can continue to play a role at woodland, reaches the long-time effect of biological Sustainable Control.
The application process of muscardine bacterium powder preparation of the present invention; The life habit of having utilized small heart-eating peach worm in soil, to survive the winter; Brought into play the characteristic that bacterial classification adapts to northern arid low temperature environment and deposits in the medium-term and long-term place of soil; Can not only cause direct control to contemporary small heart-eating peach worm larva, and can form once more the larva that survives the winter the coming year and infect.Spread fertilizer over the fields muscardine bacterium powder preparation through manual work, can increase the bacteria containing amount of soil greatly, improved the bacterial classification control effect; And have pollution-free, use conveniently, saved the tradition method of preventing and treating and be unearthed and the spent advantages such as manpower and materials of sprouting wings because of the monitoring small heart-eating peach worm.
Embodiment
Below in conjunction with embodiment the present invention is further described, but embodiment does not do any type of qualification to the present invention.
The preparation of embodiment 1 muscardine bacterium powder preparation of the present invention
(1) primary inclined plane spawn culture:
1, the preparation of slant medium:
Slant culture based component and proportioning thereof: yam 200g, glucose 20g, agar 20g, the dried worm corpse of small heart-eating peach worm larva 10g, water 1000mL.Its preparation process:
1., taking by weighing the dried worm corpse of small heart-eating peach worm larva 10g grinds adding 1000mL clear water immersion 2-3 hour.
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 filtrating to the yam back of feeling like jelly.
3., with this filtrating make up water to 1000mL, add agar 20g, boil.The dissolving back adds 20g glucose fully.Substratum is nature pH value.
4., filtrating passes through to account for 1/5 of test tube in the funnel injecting tube.The test tube mouth is with tampon and newspaper sealing.
5., test tube is with 121 ℃, 1.5pa/kg autoclaving 30min.Gone out and in time taken out behind the bacterium, while hot test tube has been put into inclined-plane (highly be about test tube 1/2).
6., treat in the test tube to put into 25 ℃ of constant incubators and to cultivate 24h after the complete cooled and solidified of substratum, do aseptic investigation.Get qualified preparation inoculation then.
2, primary inclined plane spawn culture: with preserving number is that the beauveria bassiana of CGMCC No.4526 is inoculated in the test tube that above-mentioned slant medium is housed; Put into incubator; In temperature is that 25 ℃, relative humidity are under 75% condition; Cultivated 10 days, and when treating that mycelia is covered with the inclined-plane and produces spore, changed second-class liquid isolate over to and cultivate.
(2) second-class liquid isolate is cultivated:
1, the preparation of secondary liquid nutrient medium:
Secondary liquid nutrient medium composition is formed: peptone 50g, yeast extract paste 10g, glucose 200g, water 5000mL.Its preparation process:
1., in 5000mL water, add earlier the 50g peptone, the 10g yeast extract paste, be stirred to whole dissolvings after, add 200g glucose, be divided in the 1000mL triangular flask every bottled 500mL after continuing to be stirred to its dissolving.Substratum is nature pH value.Bottleneck is with tampon and newspaper sealing.
2., with 121 ℃, 1.5pa/kg autoclaving 30min, take out sterilization back, is cooled to room temperature, can under gnotobasis, inoculate.
2, secondary liquid shaking table is cultivated: cultured primary inclined plane bacterial classification is connected substratum together scrape and put into triangular flask and cultivate; Inoculum size is 3 bottles of every test tube strains inoculations; Postvaccinal triangular flask is put into the isothermal vibration incubator; 25 ℃ of following 180r/min cultivate and covered with to the nutrient solution till the spore ball in 10 days.
(3) three grades of packed enlarged culturing of solid:
1, the preparation of solid medium in three grades of solid enlarged culturing:
Three grades of solid medium material compositions and proportioning thereof: wheat bran 35Kg, husk 14Kg, milk powder 1Kg.Its preparation process:
1., above three kinds of raw materials are weighed up in proportion after, add water by the 70%-80% of raw material gross weight, mix, up to hand-tight keep a firm hand on hold Shi Gangneng and drip till, the back that stirs is divided to pack into and is gathered in third vinyl bag.Seal sack behind Glass tubing (diameter 3cm) the insertion sack with long 10cm, bottleneck is with tampon and newspaper sealing.
2., with 121 ℃, the autoclaving 45min of 1.5pa/kg, take out sterilization back, is cooled to room temperature, can under gnotobasis, inoculate.
2, three grades of packed enlarged culturing of solid: with the cultured bacterial classification of secondary under aseptic condition; By 25 bags of every bottle of bacterial classification inoculations; Insert aseptic three grades of solid mediums are housed gather third vinyl bag (among the 30cm * 20cm); Be to cultivate 20 days under 75% the condition 25 ℃ of relative humidity, treat to cover with mycelia in the bag and spore gets final product.
(4) mensuration of spore count and germination rate
Take by weighing three grades of packed bacterial classifications of the different incubation times of 1g, put into the Erlenmeyer flask of 150ml, add sterilized water 50ml, even with magnetic stirrer.Measure spore count with blood counting chamber.
Bacteria suspension is placed 25 ℃ of cultivations, cultivate behind the 36h through microscopic examination, 5 visuals field of each rechecking, each visual field is no less than 100 spores.The statistics spore germination rate.
(5) preparation of muscardine bacterium powder preparation:
1, the filler composition in the bacterium powder preparation is formed: wheat bran 70%, husk 30%.Its preparation process:
1., above two kinds of raw materials are weighed up in proportion after, add water by the 70%-80% of raw material gross weight, mix, up to hand-tight keep a firm hand on hold Shi Gangneng and drip till, stir.
2., with 121 ℃, the autoclaving 45min of 1.5pa/kg, take out sterilization back, is cooled to room temperature.
2, the preparation of muscardine bacterium powder preparation:, under aseptic condition, stir in 1 ︰, 2 ratios with filler with three grades of cultured cultures.(among the 40cm * 25cm), every bag of weight is 2.5kg to divide the SE bag of packing into then.
The woodland of embodiment 2 muscardine preparations is used test:
1, woodland is used: in August, 2010, in fruit tree institute of Shanxi, Taigu County, Shanxi Province Academy of Agricultural Sciences jujube garden, use the bacterium powder preparation for preparing.The thick veneer of soil of 1-2cm of about 1 meter of jujube tree tree root base diameter is pushed aside, evenly spread fertilizer over the fields after muscardine bacterium powder preparation and soil are mixed thoroughly in 1 ︰, 1 ratio, and then veneer of soil is built.Consumption is the 180g/ strain.
2, deposit the place of muscardine in soil
Whenever extracted soil sample at a distance from two months in September, 2010 after woodland is used muscardine bacterium powder preparation, detect the vigor of beauveria bassiana spore in the soil, with becoming colony count and sporulation quantity to weigh.
3, effect inspection: adopt methods of sampling inspection control effect in next year, add up the worm corpse that infects muscardine death in the healthy larva of being unearthed the back of surviving the winter and the catch an illness larva and the cocoon of surviving the winter, calculate corrected mortality according to contrast again, preventive effect is at 74.0%-81.2%.The orchard borer fruit rate reduces 80.3%-90.1% then.
The woodland of embodiment 3 muscardine preparations is used test:
1, woodland is used: in August, 2010, in jujube garden, Jishan County, Shanxi Province, use the bacterium powder preparation for preparing.The thick veneer of soil of 1-2cm of about 1 meter of jujube tree tree root base diameter is pushed aside, evenly spread fertilizer over the fields after bacterium powder preparation and soil are mixed thoroughly in 1 ︰, 1 ratio, and then veneer of soil is built.Consumption is the 180g/ strain.Use muscardine bacterium powder preparation in by the end of March, 2011 according to the method described above, consumption is the 120g/ strain.
2, effect inspection: adopt methods of sampling inspection control effect in next year, add up the worm corpse that infects muscardine death in the healthy larva of being unearthed the back of surviving the winter and the catch an illness larva and the cocoon of surviving the winter, calculate corrected mortality according to contrast again, preventive effect is at 82.1%-90.5%.The orchard borer fruit rate reduces 83.8%-93.6% then.
Claims (5)
1. muscardine, its culture presevation number is CGMCCNo.4526.
2. a preparation method who prevents and treats the muscardine preparation of small heart-eating peach worm is characterized in that, comprises the steps:
(1) primary inclined plane spawn culture: with preserving number is that the beauveria bassiana bacterial classification of CGMCC No.4526 inserts and to be equipped with in the test tube of the PDA slant medium that contains healthy small heart-eating peach worm worm corpse leach liquor; Put into incubator; In temperature is that 20 ℃-28 ℃, relative humidity are to cultivate under the 70%-85% condition, treats when mycelia is covered with the inclined-plane and produces full spore subsequent use;
(2) second class inoculum is cultivated: under aseptic condition; Cultured primary inclined plane bacterial classification is scraped together with substratum; Put into the triangular flask that contains liquid nutrient medium and cultivate, inoculum size is put into the isothermal vibration incubator for 3 bottles of each test tube strains inoculations with postvaccinal triangular flask; 20 ℃-28 ℃, 150-200r/min are cultured to when covering with mycelium pellet in the nutrient solution subsequent use;
(3) three grades of solid enlarged culturing: under aseptic condition; With second class inoculum; Be 1 by volume: the inoculum size of 10-25 inserts in the solid medium of the bacterium of going out; In temperature is that 20 ℃-28 ℃, relative humidity are to cultivate under the 70%-85% condition, to substratum, covers with white hypha, promptly gets three grades of cultures;
(4) bacterium powder preparation preparation: with raw materials by weight meter wheat bran 60-80, husk 20-40 (preferred: wheat bran 70, husk 30) mixing, and add water 60%-80% by the raw material gross weight and stir, sterilization, be cooled to room temperature after, promptly get filler; With filler and three grades of cultures in 1-3: 1 ratio stirs in sterilisable chamber, and packing promptly gets muscardine bacterium powder preparation;
Described liquid nutrient medium is the SDAY substratum;
Described solid medium makes through following method: by weight percentage, with raw material wheat bran 68-72, husk 26-30, milk powder 1-4 mixing, and the water of adding raw material gross weight 60%-80% stir, and sterilization makes.
3. a kind of preparation method who prevents and treats the muscardine preparation of small heart-eating peach worm as claimed in claim 2; It is characterized in that; The described PDA slant medium proportioning that contains healthy small heart-eating peach worm worm corpse leach liquor is: yam 200g/L; Glucose 20g/L, agar 20g/L, the dried worm corpse of small heart-eating peach worm larva 10g/L.
4. a kind of preparation method who prevents and treats the muscardine preparation of small heart-eating peach worm as claimed in claim 2 is characterized in that raw material by weight percentage in the described solid medium: wheat bran 70, husk 28, milk powder 2.
5. the application process of the muscardine preparation that makes like the said preparation method of claim 2: the thick veneer of soil of 1-2cm peripheral about 1 meter of the tree root base portion that small heart-eating peach worm is taken place is pushed aside; Evenly spread fertilizer over the fields after muscardine bacterium powder preparation and soil mixed thoroughly in 1 ︰ 0.5-1.5 ratio, and then veneer of soil built get final product; Consumption is the 150-250g/ strain, time of application be small heart-eating peach worm will take off the fruit bury survive the winter before.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN108378063A (en) * | 2018-05-18 | 2018-08-10 | 中国农业科学院农业环境与可持续发展研究所 | A kind of preparation method of muscardine granule |
| CN110317770A (en) * | 2019-08-15 | 2019-10-11 | 贵州正春林业科技服务有限公司 | A kind of extracting method of beauveria bassiana spore |
| CN113200793A (en) * | 2021-04-22 | 2021-08-03 | 陕西科技大学 | Agricultural humic acid nutrient with biological control function and preparation method and application thereof |
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| CN1823593A (en) * | 2006-01-10 | 2006-08-30 | 山西大学 | Beauveria bassiana preparation used for preventing and controlling pine moth, its preparation method and use |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108378063A (en) * | 2018-05-18 | 2018-08-10 | 中国农业科学院农业环境与可持续发展研究所 | A kind of preparation method of muscardine granule |
| CN110317770A (en) * | 2019-08-15 | 2019-10-11 | 贵州正春林业科技服务有限公司 | A kind of extracting method of beauveria bassiana spore |
| CN113200793A (en) * | 2021-04-22 | 2021-08-03 | 陕西科技大学 | Agricultural humic acid nutrient with biological control function and preparation method and application thereof |
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Application publication date: 20120926 |