CN102321543A - Carbohydrate protective agent for increasing heat resistance of beauveria bassiana spore - Google Patents
Carbohydrate protective agent for increasing heat resistance of beauveria bassiana spore Download PDFInfo
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- CN102321543A CN102321543A CN201110252924A CN201110252924A CN102321543A CN 102321543 A CN102321543 A CN 102321543A CN 201110252924 A CN201110252924 A CN 201110252924A CN 201110252924 A CN201110252924 A CN 201110252924A CN 102321543 A CN102321543 A CN 102321543A
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Abstract
The invention discloses a carbohydrate protective agent for increasing heat resistance of beauveria bassiana spores, and is applicable to the fields of preparation, preservation and field application of beauveria bassiana spore preparations; the protective agent particularly comprises 5%-13% of disaccharides, tween-80 with a final concentration of 0.01%-0.1%, and the balance of water. Beauveria bassiana spores can be used to control tea leafhoppers; the heat resistance of the spores is increased; the heat damage of the spores during the preparation and preservation of the preparation is reduced; the resistance of beauveria bassiana spores to ambient high temperatures is increased during field application; thus the survival rate and the infection rate of beauveria bassiana spores are increased; and the effect of biological control is increased.
Description
Technical field
The present invention relates to a kind of carbohydrate protective material that improves beauveria bassiana spore heat impedance, can be used for fields such as beauveria bassiana spore formulation preparation, preservation and field application.
Background technology
Along with the growing interest of the mankind, widely popularize the new trend that biological pesticide has become global agricultural chemicals industry development to environment protection and self health.Muscardine is as a kind of broad spectrum pathogenic fungi, is easy to produce because of it has, host range is wide, the phase of infecting is long, harm people and other vertebratess and valuable advantages such as popular potentiality do not become and study and use at present maximum one of microbial pesticides that are in the world.
Bacterial strain (beauveria bassiana CCTCC No:M209238) used among the present invention is to the No.1 insect of present tea tree---and " artificial eye unit cell smaller green leaf hopper " has remarkable control effect.Yet the beauveria bassiana spore preparation all can cause the active reduction of spore even dead because of receiving thermal damage in preparation, preservation and use.Wherein, the heat in the preparation process is come self-desiccation, and the heat in the preserving process uses the heat in (field application) process to generate heat from solar radiation or host's behavior from the preservation environment.Therefore, improve the heat impedance of beauveria bassiana spore, to improving its killing rate and the biological control effect is most important.
Summary of the invention
Technical problem to be solved by this invention provides a kind of carbohydrate protective material that improves beauveria bassiana spore heat impedance.
For solving the problems of the technologies described above, said protective material is formed as follows: the disaccharides of 5%-13%, final concentration are the tween-80 of 0.01%-0.1%, and all the other are water.
Said beauveria bassiana (Beauveria bassiana) is preserved in Chinese typical culture collection center; Deposit number is CCTCC No:M209238; This bacterial strain information is open in Chinese patent 200910264664.8, date of publication on June 9th, 2010, publication No. CN101724571A.
The protectant method of use of the spore of said beauveria bassiana is: is that the ratio of 5%-13% is added in the spore suspension of beauveria bassiana with said disaccharides protective material in mass volume ratio.
Said disaccharides is any in sucrose, SANMALT-S, trehalose or the lactose.Beauveria bassiana spore suspension preferred preparation method is following: get an amount of disaccharides in the 25mL test tube, add the tween-80 solution of 9.5mL 0.05%, boiling water bath also is stirred to carbohydrate and dissolves fully, after the ice-water bath cooling, adds 0.5mL 2 * 10
9The spore suspension of spore/mL, and with vibrator with its mixing.
The detection method of spore heat impedance: 1) above-mentioned spore suspension uniform mixing liquid is placed 45 ℃ of thermostat water baths, behind the water-bath 10min (concussion simultaneously), take out immediately, place the cooling of mixture of ice and water ice bath; 2) cooled mixed liquor is transferred in the 250mL triangular flask, added 40mL 2.5% wheat bran leach liquor again, mixing places 25 ℃, cultivates 12h in the 180r/min shaking table; 3) draw a nutrient solution with the glue head straw, drip on the blood counting chamber, and in detecting and calculate its spore germination rate (when germ tube length was not less than the spore diameter, this spore was regarded as sprouting) under the electron microscope.
Beneficial effect of the present invention is following: the beauveria bassiana spore can be used for preventing and treating the tealeaves smaller green leaf hopper; The raising of its spore heat impedance; Reduced the thermal damage of spore in formulation preparation and preserving process; When also having increased the beauveria bassiana spore and having used in the field to environment in the pyritous resistivity, thereby improved the survival rate and the infection rate of beauveria bassiana spore, and then improve the effect of its biological control.
Embodiment
Embodiment 1: the preparation of spore suspension and nutritive medium
The preparation of spore suspension:
1) wash-out.The used bacterial classification of the present invention is beauveria bassiana Beauveria bassiana (deposit number is CCTCC No:M209238); Get the proper amount of fresh fermenting culture in the triangular flask of 250mL; Add an amount of 0.05% tween-80 solution and an amount of granulated glass sphere; With glass stick culture is smashed to pieces, with the vibrator 10min at least that vibrates at a high speed.
2) filter.After the vibration, use the layer 2-4 filtered through gauze, collect filtrating.
3) centrifugal.It is centrifugal to filtrate, and condition is: 8000r/min, 5min, 4 ℃, supernatant discarded.
4) redissolve.Throw out after centrifugal is transferred in the 50mL centrifuge tube, added the tween-80 solution and an amount of granulated glass sphere of 40mL 0.05%, shake 20min at least at a high speed with vibrator.
5) counting.Get the spore suspension after the above-mentioned redissolution of 1mL, the tween-80 solution dilution 50-100 with 0.05% doubly after, count with blood counting chamber.
6) dilution.Behind the counting, with protospore suspension-s with 0.05% tween-80 solution dilution to 2 * 10
9Spore/mL, for use.
The preparation of wheat bran leach liquor (nutritive medium):
Take by weighing the 25g wheat bran in pot, add in the 1000mL zero(ppm) water, boil and stir; Keep the boiling 30min (more than); The layer 2-4 filtered through gauze is used in the cooling back, collects filtrating, with zero(ppm) water filtrating is complemented to 1000mL again; It is 2.5% wheat bran leach liquor that gained solution is concentration, also is to cultivate the used nutritive medium of spore suspension.115 ℃, the 15min sterilization, for use.
Comparative Examples:
Contrast 1: with 10mL 1 * 10
8Spore/mL spore suspension places the 250mL triangular flask, adds 40mL 2.5% wheat bran leach liquor again, and mixing places 25 ℃, cultivates 12h in the 180r/min shaking table; Draw a nutrient solution with the glue head straw, drip on the blood counting chamber, and in detecting and calculate its spore germination rate (when germ tube length was not less than the spore diameter, this spore was regarded as sprouting) under the electron microscope.
Contrast 2: with 10mL 1 * 10
8Spore/mL spore suspension places the 250mL triangular flask, add 40mL 2.5% wheat bran leach liquor again and with the 5g sucrose dissolved in mixed solution, mixing places 25 ℃, cultivates 12h in the 180r/min shaking table; Draw a nutrient solution with the glue head straw, drip on the blood counting chamber, and in detecting and calculate its spore germination rate (when germ tube length was not less than the spore diameter, this spore was regarded as sprouting) under the electron microscope.
Contrast 3: with 10mL 1 * 10
8Spore/mL spore suspension places the 250mL triangular flask, adds 40mL 2.5% wheat bran leach liquor again and 5g SANMALT-S is dissolved in the mixed solution, and mixing places 25 ℃, cultivates 12h in the 180r/min shaking table; Draw a nutrient solution with the glue head straw, drip on the blood counting chamber, and in detecting and calculate its spore germination rate (when germ tube length was not less than the spore diameter, this spore was regarded as sprouting) under the electron microscope.
Contrast 4: with 10mL 1 * 10
8Spore/mL spore suspension places the 250mL triangular flask, adds 40mL 2.5% wheat bran leach liquor again and the 5g trehalose is dissolved in the mixed solution, and mixing places 25 ℃, cultivates 12h in the 180r/min shaking table; Draw a nutrient solution with the glue head straw, drip on the blood counting chamber, and in detecting and calculate its spore germination rate (when germ tube length was not less than the spore diameter, this spore was regarded as sprouting) under the electron microscope.
Contrast 5: with 10mL 1 * 10
8Spore/mL spore suspension places the 250mL triangular flask, adds 40mL 2.5% wheat bran leach liquor again and the 5g lactose is dissolved in the mixed solution, and mixing places 25 ℃, cultivates 12h in the 180r/min shaking table; Draw a nutrient solution with the glue head straw, drip on the blood counting chamber, and in detecting and calculate its spore germination rate (when germ tube length was not less than the spore diameter, this spore was regarded as sprouting) under the electron microscope.
The result sees table 1
Table 1
Result in the table 1 shows that sucrose, SANMALT-S, trehalose and lactose all do not have obvious facilitation to the sprouting of beauveria bassiana spore.
Embodiment 2: sucrose
The protectant spore suspension uniform mixing of above-mentioned interpolation sucrose liquid is placed 45 ℃ of thermostat water baths, behind the water-bath 10min (concussion simultaneously), take out immediately, place the cooling of mixture of ice and water ice bath; Cooled mixed liquor is transferred in the 250mL triangular flask, added 40mL 2.5% wheat bran leach liquor again, mixing places 25 ℃, cultivates 12h in the 180r/min shaking table; Draw a nutrient solution with the glue head straw, drip on the blood counting chamber, and in detecting and calculate its spore germination rate (when germ tube length was not less than the spore diameter, this spore was regarded as sprouting) under the electron microscope, the result is as shown in table 2.
Table 2
Embodiment 3: SANMALT-S
The protectant spore suspension uniform mixing of above-mentioned interpolation SANMALT-S liquid is placed 45 ℃ of thermostat water baths, behind the water-bath 10min (concussion simultaneously), take out immediately, place the cooling of mixture of ice and water ice bath; Cooled mixed liquor is transferred in the 250mL triangular flask, added 40mL 2.5% wheat bran leach liquor again, mixing places 25 ℃, cultivates 12h in the 180r/min shaking table; Draw a nutrient solution with the glue head straw, drip on the blood counting chamber, and in detecting and calculate its spore germination rate (when germ tube length was not less than the spore diameter, this spore was regarded as sprouting) under the electron microscope, the result is as shown in table 3.
Table 3
Embodiment 4: trehalose
The protectant spore suspension uniform mixing of above-mentioned interpolation trehalose liquid is placed 45 ℃ of thermostat water baths, behind the water-bath 10min (concussion simultaneously), take out immediately, place the cooling of mixture of ice and water ice bath; Cooled mixed liquor is transferred in the 250mL triangular flask, added 40mL 2.5% wheat bran leach liquor again, mixing places 25 ℃, cultivates 12h in the 180r/min shaking table; Draw a nutrient solution with the glue head straw, drip on the blood counting chamber, and in detecting and calculate its spore germination rate (when germ tube length was not less than the spore diameter, this spore was regarded as sprouting) under the electron microscope, the result is as shown in table 4.
Table 4
Embodiment 5: lactose
The protectant spore suspension uniform mixing of above-mentioned interpolation lactose liquid is placed 45 ℃ of thermostat water baths, behind the water-bath 10min (concussion simultaneously), take out immediately, place the cooling of mixture of ice and water ice bath; Cooled mixed liquor is transferred in the 250mL triangular flask, added 40mL 2.5% wheat bran leach liquor again, mixing places 25 ℃, cultivates 12h in the 180r/min shaking table; Draw a nutrient solution with the glue head straw, drip on the blood counting chamber, and in detecting and calculate its spore germination rate (when germ tube length was not less than the spore diameter, this spore was regarded as sprouting) under the electron microscope, the result is as shown in table 5.
Table 4
Claims (3)
1. a carbohydrate protective material that improves beauveria bassiana spore heat impedance is characterized in that, the concrete composition as follows: the disaccharides of 5%-13%, final concentration are the tween-80 of 0.01%-0.1%, and all the other are water.
2. according to the said protective material of claim 1, it is characterized in that said disaccharides is any in sucrose, SANMALT-S, trehalose or the lactose.
3. according to the said protective material of claim 3; It is characterized in that; Protectant addition means is following in the beauveria bassiana spore suspension: get an amount of disaccharides in the 25mL test tube, add the tween-80 solution of 9.5mL 0.05%, boiling water bath also is stirred to carbohydrate and dissolves fully; After the ice-water bath cooling, add 0.5mL 2 * 10
9The spore suspension of spore/mL, and with vibrator with its mixing.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667062A (en) * | 2013-12-13 | 2014-03-26 | 江南大学 | Protective agent for low-temperature preservation of asexual spores of antrodia cinnamomea and protective agent using method |
| CN107258823A (en) * | 2017-06-14 | 2017-10-20 | 齐鲁工业大学 | A kind of beauveria bassiana granular formulation preparation method and applications of control of grubs |
| CN110184197A (en) * | 2019-05-15 | 2019-08-30 | 云南星耀生物制品有限公司 | A kind of beauveria bassiana oil-suspending agent |
| CN111657275A (en) * | 2020-07-20 | 2020-09-15 | 北京嘉景生物科技有限责任公司 | Insecticidal fungal spore aqueous suspension and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724571A (en) * | 2009-12-21 | 2010-06-09 | 无锡益鹏集团有限公司 | Method for increasing yield of solid fermentation beauveria bassiana spores by adding nutrient substances |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724571A (en) * | 2009-12-21 | 2010-06-09 | 无锡益鹏集团有限公司 | Method for increasing yield of solid fermentation beauveria bassiana spores by adding nutrient substances |
Non-Patent Citations (4)
| Title |
|---|
| QIAN LIU等: "Physiological implication of intracellular trehalose and mannitol changes in response of entomopathogenic fungus Beauveria bassiana to thermal stress", 《ANTONIE VAN LEEUWENHOEK》, vol. 95, 12 October 2008 (2008-10-12), pages 73 * |
| 任巧云等: "不同浓度吐温-80与白僵菌分生孢子的相容性测定", 《安徽农业科学》, vol. 37, no. 18, 20 June 2009 (2009-06-20), pages 8555 - 8687 * |
| 徐均焕等: "球孢白僵菌孢子萌发促进剂的筛选及其对杀蚜效果的增强作用", 《植物保护学报》, vol. 30, no. 1, 15 February 2003 (2003-02-15), pages 45 - 50 * |
| 李鸿文: "球孢白僵菌不同菌株分生孢子耐热力及相关孢壁蛋白分析", 《中国优秀硕士学位论文全文数据库农业科技辑》, 15 October 2007 (2007-10-15) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667062A (en) * | 2013-12-13 | 2014-03-26 | 江南大学 | Protective agent for low-temperature preservation of asexual spores of antrodia cinnamomea and protective agent using method |
| CN103667062B (en) * | 2013-12-13 | 2016-06-15 | 江南大学 | A kind of Antrodia camphorata asexual spore low-temperature preservation protective agent and using method thereof |
| CN107258823A (en) * | 2017-06-14 | 2017-10-20 | 齐鲁工业大学 | A kind of beauveria bassiana granular formulation preparation method and applications of control of grubs |
| CN110184197A (en) * | 2019-05-15 | 2019-08-30 | 云南星耀生物制品有限公司 | A kind of beauveria bassiana oil-suspending agent |
| CN110184197B (en) * | 2019-05-15 | 2020-04-03 | 云南星耀生物制品有限公司 | Beauveria bassiana oil suspending agent |
| CN111657275A (en) * | 2020-07-20 | 2020-09-15 | 北京嘉景生物科技有限责任公司 | Insecticidal fungal spore aqueous suspension and preparation method thereof |
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| CN102321543B (en) | 2013-10-16 |
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