CN101024816A - Method for preparing Trichderma bacteria conididium powder and use thereof - Google Patents
Method for preparing Trichderma bacteria conididium powder and use thereof Download PDFInfo
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Abstract
The invention discloses a manufacturing method and the application for a wood fungus conidium dust agent. It gains wood fungus from separating leaf of vegetables, purifying and transplanting to PDA test tube bevel to be used as mother fungus, adjusting temperature, pH value, and oxygen supplying quantity to produce spore liquid, and gaining large quantity of dry proofing conidium by adopting solid fermenting, mixing crashed condium and superfine diatomite to form wood fungus conidium dust agent. The method could be used to prevent vegetable producing.
Description
Technical field
The present invention relates to a kind of biocontrol fungi that is used to prevent and treat the greenhouse leaf diseases---the production method of Trichoderma (Trichoderma viride) bacteria conididium powder, the preparation method and the application thereof of the novel biological prevention and control agent of definite a kind of superparasitism fungi of saying so.
Background technology
Along with the fast development of protection ground cultivated area, single cropping all the year round such as vegetables that economic worth is higher such as cucumber, tomato, capsicum, disease takes place more and more heavier, and especially gray mold etc. often causes underproduction 20-30%, even total crop failure.The vegetable disease control of protection ground is controlled disease by frequent dispenser, not only the cost height always based on chemoprevention; time-consuming, and pesticide residue height in the vegetables, promptly be detrimental to health; influence foreign exchange earning again, therefore increasing poisonous chemical pesticide is limited to use gradually.At present, all in the research and development dynamics of increasing biological pesticide, develop countries in the world, exploitation is nontoxic to people, animal, and the microbial pesticide of environmentally safe has become focus.The antagonism fungi that Trichoderma has important biological and ecological methods to prevent plant disease, pests, and erosion to be worth as a class, as far back as 20th century the '30s just have been found that plant pathogenic fungi is had antagonistic action, clear and definite now its mechanism of action comprises microbiotic, hyperparasitism, bacteriolysis, the competition effect etc. of producing.Competition of Trichoderma intensive and viability play an important role in the process that suppresses pathogenic bacteria, are a kind of biological and ecological methods to prevent plant disease, pests, and erosion bacterial classifications that value of exploiting and utilizing is arranged very much.Having developed registration in the world has the Trichoderma product, and also there is the patent of some Trichodermas in China, but very few as the preparation that can really use on producing.Existing most of Trichoderma research all is used to prevent and treat soil-borne disease of crop, and existing research about Trichoderma control leaf diseases mostly is the conidium aqua.Facts have proved, be difficult to obtain pure Trichoderma conidium by liquid submerged fermentation, often mixed have many chlamydospores and mycelia and microbial inoculum can not normally be sprayed, even spraying can be carried out, the conidium resistance that exists liquid fermenting to produce again is poor, be difficult for field planting at the crop body surface, problems such as the conidium aqua storage time weak point of making.The method of employing spraying simultaneously increases the humidity in the greenhouse again, and high humidity is a greenhouse leaf diseases popular key factor, has restricted the application of Trichoderma aspect control plant leaf diseases to a great extent.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and application thereof that is used to prevent and treat the Trichoderma conidium biological control dust agent of greenhouse vegetable leaf diseases (gray mold, leaf mold etc.), cause to overcome conventional spray method that humidity increases to preventing and treating the disadvantageous effect that disease is brought in the greenhouse.
The objective of the invention is to realize by following content:
The biocontrol microorganisms that is adopted is taken from the Trichoderma of vegetable crop leaf table, be transferred to after purified and make female microbial inoculum in the test tube slant, shaking culture is a liquid spawn in triangular flask, makes it produce conidium through solid fermentation again, this conidium with make bacteria conididium powder after the ultrafine carrier mixes.Small and light with the Trichoderma conidium that present method is produced, anti-drying, degeneration-resistant border.
Described trichderma bacteria conididium powder is prepared from by following technology processing process:
1) the female strain preparation in inclined-plane: get vegetables blade 5-10 gram, add sterilized water 50-100 milliliter, after vibration, add water and become 1000-10000 times of diluent; Get potato 150-200 gram, glucose 15-20 gram, rose-bengal 0.04-0.07 gram, Vetstrep 0.03-0.05 gram, agar 17-20 gram, 1000 milliliters in water is mixed with female bacterium culture medium; Pour into after getting diluent and bacterium culture medium mixing with 1: 9 and make culture medium flat plate in the culture dish, cultivated 48 hours down, after separation obtains the Trichoderma bacterial strain, be purified, be transferred to and make the female bacterial classification in inclined-plane in the test tube slant at 25-28 ℃;
2) second class inoculum preparation: get potato 150-200 gram, glucose or sucrose 15-20 gram, potassium primary phosphate 1-2 gram, sal epsom 0.5-1 gram, 1000 milliliters in water is mixed with the second order fermentation nutrient solution, adjust pH is 6-8, is divided in the 250-1000 milliliter triangular flask, and liquid amount is the 100-300 milliliter, sterilized 20-40 minute down at 121 ℃, in sterilisable chamber the female bacterial classification in inclined-plane changed over to and shake bottle, shaking speed is 150-200 rev/min, leavening temperature 28-31 ℃, fermentation time is 3-5 days, obtains second class inoculum;
3) solid fermentation: the solid fermentation medium component that is adopted is by weight percentage: culture material is wheat bran or straw powder or Semen Maydis grit 70-80%, rice husk 20-30%, water is the 50-100% of culture material, making method is to pack in high-temperature resistance plastice bag or the fermentation flask making solid medium behind culture material and the water thorough mixing, sterilized 40-60 minute down at 121 ℃, insert second class inoculum, connect the bacterium amount and be the 5-10% of solid medium, leavening temperature is 25-30 ℃, connecing behind the bacterium 3-5 days stirs once, fermentation time 7-10 days, becoming green to culture material was the Trichoderma conidium;
4) production of trichderma bacteria conididium powder: will be fully loaded with the conidial solid culture medium of Trichoderma after the normal temperature drying, be ground into the female powder of Trichoderma conidium with Universalpulverizer, make bacteria conididium powder after getting the female powder of 5-10% conidium, 5-10%300-350 order light calcium carbonate, 80-90%300-350 order diatomite thorough mixing by weight percentage.
Trichderma bacteria conididium powder is sprayed control canopy dish leaf diseases in plastic greenhouse and various greenhouse by powder sprayer.
The invention has the advantages that: small and light according to the Trichoderma conidium that technical parameter provided by the present invention obtains, easily fly away anti-drying, anti-storage.The Trichoderma conidium is the same floating in the booth space to " cigarette " by powder sprayer, can be deposited to each level of plant, and can deposit and be attached to the comparison concealment part, each places such as canopy wall, column, lack than conventional spore spray method spraying times, consumption is little, the preventive effect height, easy to operate, and do not influence other farming operation after dusting.Can reduce simultaneously the humidity in the greenhouse (plastic greenhouse), make floral organ and blade face not tie water or tie water less, the Trichoderma spore by reveal morning and the greenhouse in sooner or later humidity can sprout, breed, reach superparasitism purpose to pathogenic bacteria.With every 667m
2Medication 0.5-1 kilogram, leaf diseases such as control greenhouse vegetable gray mold, leaf mold, prevention effect reaches 60%-85%.
The present invention is applicable to most of trichoderma strains, and method is simple, and the water and electric saving is suitable for different scales production, and market outlook are wide.
Embodiment
Embodiment 1
Preparation technology's flow process of trichderma bacteria conididium powder is as follows:
1. slant strains preparation
Get tomato leaf 5 grams, add 50 milliliters of sterilized waters, thin up becomes 1000 times of diluents after vibrating; Get potato 150 grams, glucose 20 grams, rose-bengal 0.07 gram, Vetstrep 0.03 gram, agar 17 grams, 1000 milliliters in water is made into female bacterium culture medium.Making method is: will be cut into the fritter of 1 centimeter square behind the peeling potatoes, adding 1000 ml waters boiled 30 minutes, with gauze elimination potato ball, add agar, add water and supply 1000 milliliters, add glucose and rose-bengal then and make it to dissolve fully after pack into triangular flask sterilization, 121 ℃ of 30 sterilizations minute down, fall and add Vetstrep before dull and stereotyped.Get diluent and mix to pour into 1: 9 part by weight with female bacterium culture medium and make culture medium flat plate in the culture dish, cultivated 48 hours down, obtain the Trichoderma bacterial strain, be transferred to after purified and make female bacterial classification in the test tube slant through separating at 25 ℃.Its test tube slant substratum is conventional PDA substratum (1000 milliliters of potato 200 gram, glucose 20 grams, agar 20 grams, water).
2. second class inoculum preparation
Get potato 150 grams, glucose 15 grams, potassium primary phosphate 1 gram, sal epsom 0.5 gram, 1000 milliliters in water is mixed with the second order fermentation nutrient solution.Making method is: will be cut into the fritter of 1 centimeter square behind the peeling potatoes, adding 1000 ml waters boiled 20 minutes, with gauze elimination potato ball, add water and supply 1000 milliliters, after adding glucose or sucrose then and making it to dissolve fully, adjust pH is 6, is divided in 250 milliliters of triangular flasks, liquid amount is 100 milliliters, sterilizes 20 minutes down at 121 ℃.In sterilisable chamber the female bacterial classification in inclined-plane changed over to and shake bottle, shaking speed is 170 rev/mins, 28 ℃ of leavening temperatures, and fermentation time is 3 days, obtains second class inoculum one conidium liquid.
3. solid fermentation
The solid fermentation medium component that is adopted is by weight percentage: culture material is straw powder 80%, rice husk 20%, and water is 100% of culture material.Making method is: pack in the high-temperature resistance plastice bag making solid medium behind culture material and the water thorough mixing, sterilized 40 minutes down at 121 ℃, insert second class inoculum, connect the bacterium amount and be 10% of substratum weight, leavening temperature is 25 ℃, connect behind the bacterium the 3rd day and stir once, it is anti-dry Trichoderma conidium that fermentation time 7 days, culture material become green.
4. the production of trichderma bacteria conididium powder
To be fully loaded with the conidial solid culture medium of Trichoderma after the normal temperature drying, be ground into the female powder of Trichoderma conidium with Universalpulverizer, make bacteria conididium powder after getting the female powder of 10% conidium, 10%300 order light calcium carbonates, 80%350 order diatomite thorough mixing by weight percentage.
With trichderma bacteria conididium powder with the every 667m of Farm powder sprayer
2The greenhouse dusts 1 kilogram, and the control graw mold of tomato is used at premorbid or their early stage, and prevention effect reaches more than 80%.
Embodiment 2
1. slant strains preparation
Get cucumber leaves 8 grams, add 80 milliliters of sterilized waters, thin up becomes 8000 times of diluents after vibrating; Get potato 180 grams, glucose 18 grams, rose-bengal 0.05 gram, Vetstrep 0.04 gram, agar 18 grams, 1000 milliliters in water is made into female bacterium culture medium.Making method is: will be cut into the fritter of 1 centimeter square behind the peeling potatoes, adding 1000 ml waters boiled 30 minutes, with gauze elimination potato ball, add agar, add water and supply 1000 milliliters, add glucose and rose-bengal then and make it to dissolve fully after pack into triangular flask sterilization, 121 ℃ of 30 sterilizations minute down, fall and add Vetstrep before dull and stereotyped.Get diluent and mix at 1: 9 to pour into part by weight with female bacterium culture medium and make culture medium flat plate in the culture dish, cultivated 48 hours down, obtain the Trichoderma bacterial strain, be transferred to after purified and make female bacterial classification in the test tube slant through separating at 26 ℃.Its test tube slant substratum is conventional PDA substratum (1000 milliliters of potato 200 gram, glucose 20 grams, agar 20 grams, water).
2. second class inoculum preparation
Get potato 180 grams, glucose 18 grams, potassium primary phosphate 1.5 grams, sal epsom 0.8 gram, 1000 milliliters in water is mixed with the second order fermentation nutrient solution.Making method is: will be cut into the fritter of 1 centimeter square behind the peeling potatoes, adding 1000 ml waters boiled 20 minutes, with gauze elimination potato ball, add water and supply 1000 milliliters, after adding glucose or sucrose then and making it to dissolve fully, adjust pH is 7, is divided in 500 milliliters of triangular flasks, liquid amount is 200 milliliters, sterilizes 30 minutes down at 121 ℃.In sterilisable chamber the female bacterial classification in inclined-plane changed over to and shake bottle, shaking speed is 180 rev/mins, 29 ℃ of leavening temperatures, fermentation time is 4 days, obtain second class inoculum-. conidium liquid.
3. solid fermentation
The solid fermentation medium component that is adopted is by weight percentage: culture material is wheat bran 75%, rice husk 25%, and water is 80% of culture material.Making method is: pack in the high-temperature resistance plastice bag making solid medium behind culture material and the water thorough mixing, sterilized 50 minutes down at 121 ℃, insert second class inoculum, connect the bacterium amount and be 8% of substratum weight, leavening temperature is 28 ℃, connect behind the bacterium the 4th day and stir once, it is anti-dry Trichoderma conidium that fermentation time 8 days, culture material become green.
4. the production of trichderma bacteria conididium powder
To be fully loaded with the conidial solid culture medium of Trichoderma after the normal temperature drying, be ground into the female powder of Trichoderma conidium with Universalpulverizer, make bacteria conididium powder after getting the female powder of 5% conidium, 10%325 order light calcium carbonates, 85%300 order diatomite thorough mixing by weight percentage.
With trichderma bacteria conididium powder with the every 667m of Farm powder sprayer
2The greenhouse dusts 0.5 kilogram, and the control gray mold of cucumber is used at premorbid or their early stage, and prevention effect reaches more than 75%.
Embodiment 3
1. slant strains preparation
Get tomato leaf 10 grams, add 100 milliliters of sterilized waters, thin up becomes 10000 times of diluents after vibrating, and gets potato 200 grams, glucose 15 grams, and rose-bengal 0.04 gram, Vetstrep 0.05 gram, agar 20 grams, 1000 milliliters in water is made into female bacterium culture medium.Making method is: will be cut into the fritter of 1 centimeter square behind the peeling potatoes, adding 1000 ml waters boiled 30 minutes, with gauze elimination potato ball, add agar, add water and supply 1000 milliliters, add glucose and rose-bengal then and make it to dissolve fully after pack into triangular flask sterilization, 121 ℃ of 30 sterilizations minute down, fall and add Vetstrep before dull and stereotyped.Get diluent and mix at 1: 9 to pour into part by weight with female bacterium culture medium and make culture medium flat plate in the culture dish, cultivated 48 hours down, obtain the Trichoderma bacterial strain, be transferred to after purified and make female bacterial classification in the test tube slant through separating at 28 ℃.Its test tube slant substratum is conventional PDA substratum (1000 milliliters of potato 200 gram, glucose 20 grams, agar 20 grams, water).
2. second class inoculum preparation
Get potato 200 grams, sucrose 20 grams, potassium primary phosphate 2 grams, sal epsom 1 gram, 1000 milliliters in water is mixed with the second order fermentation nutrient solution.Making method is: will be cut into the fritter of 1 centimeter square behind the peeling potatoes, add 1000 ml waters and boiled 30 minutes, with gauze elimination potato ball, add water and supply 1000 milliliters, after adding sucrose then and making it to dissolve fully, adjust pH is 8, is divided in 1000 milliliters of triangular flasks.Liquid amount is 300 milliliters, 121 ℃ of down sterilizations 40 minutes, in sterilisable chamber the female bacterial classification in inclined-plane is changed over to and shakes bottle, and shaking speed is 200 rev/mins, and 31 ℃ of leavening temperatures, fermentation time are 10 days, obtain second class inoculum-conidium liquid.
3. solid fermentation
The solid fermentation medium component that is adopted is by weight percentage: culture material is Semen Maydis grit 70%, rice husk 30%, and water is 50% of culture material.Making method is: pack in the high temperature resistant fermentation flask making solid medium behind culture material and the water thorough mixing, sterilized 60 minutes down at 121 ℃, insert second class inoculum, connect the bacterium amount and be 5% of substratum weight, leavening temperature is 30 ℃, connect behind the bacterium the 5th day and rock once, it is anti-dry Trichoderma conidium that fermentation time 10 days, culture material become green.
4. the production of trichderma bacteria conididium powder
To be fully loaded with the conidial solid culture medium of Trichoderma after the normal temperature drying, be ground into the female powder of Trichoderma conidium with Universalpulverizer, make bacteria conididium powder after getting the female powder of 5% conidium, 5%350 order light calcium carbonates, 90%325 order diatomite thorough mixing by weight percentage.
With Trichoderma spore dust agent with the every 667m of Farm powder sprayer
2The greenhouse dusts 0.8 kilogram, prevents and treats leaf muld of tomato, uses at premorbid or their early stage, and prevention effect reaches more than 60%.
Claims (2)
1. the production method of a trichderma bacteria conididium powder is characterized in that comprising following concrete steps:
1) the female strain preparation in inclined-plane: get vegetables blade 5-10 gram, add sterilized water 50-100 milliliter, after vibration, add water and become 1000-10000 times of diluent; Get potato 150-200 gram, glucose 15-20 gram, rose-bengal 0.04-0.07 gram, Vetstrep 0.03-0.05 gram, agar 17-20 gram, 1000 milliliters in water is mixed with female bacterium culture medium; Pour into after getting diluent and bacterium culture medium mixing with 1: 9 and make culture medium flat plate in the culture dish, cultivated 48 hours down, after separation obtains the Trichoderma bacterial strain, be purified, be transferred to and make the female bacterial classification in inclined-plane in the test tube slant at 25-28 ℃;
2) second class inoculum preparation: get potato 150-200 gram, glucose or sucrose 15-20 gram, potassium primary phosphate 1-2 gram, sal epsom 0.5-1 gram, 1000 milliliters in water is mixed with the second order fermentation nutrient solution, adjust pH is 6-8, is divided in the 250-1000 milliliter triangular flask, and liquid amount is the 100-300 milliliter, sterilized 20-40 minute down at 121 ℃, in sterilisable chamber the female bacterial classification in inclined-plane changed over to and shake bottle, shaking speed is 150-200 rev/min, leavening temperature 28-31 ℃, fermentation time is 3-5 days, obtains second class inoculum;
3) solid fermentation: the solid fermentation medium component that is adopted is by weight percentage: culture material is wheat bran or straw powder or Semen Maydis grit 70-80%, rice husk 20-30%, water is the 50-100% of culture material, making method is to pack in high-temperature resistance plastice bag or the fermentation flask making solid medium behind culture material and the water thorough mixing, sterilized 40-60 minute down at 121 ℃, insert second class inoculum, connect the bacterium amount and be the 5-10% of solid medium, leavening temperature is 25-30 ℃, connecing behind the bacterium 3-5 days stirs once, fermentation time 7-10 days, becoming green to culture material was the Trichoderma conidium;
4) production of trichderma bacteria conididium powder: will be fully loaded with the conidial solid culture medium of Trichoderma after the normal temperature drying, be ground into the female powder of Trichoderma conidium with Universalpulverizer, make bacteria conididium powder after getting the female powder of 5-10% conidium, 5-10%300-350 order light calcium carbonate, 80-90%300-350 order diatomite thorough mixing by weight percentage.
2. application of trichderma bacteria conididium powder according to claim 1 is characterized in that trichderma bacteria conididium powder is sprayed control canopy dish leaf diseases in plastic greenhouse and various greenhouse by powder sprayer.
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| CN101874484A (en) * | 2009-12-18 | 2010-11-03 | 江苏耕耘化学有限公司 | Paste dosage-form trichoderma biopesticide composite and preparation method thereof |
| CN102067885A (en) * | 2010-03-31 | 2011-05-25 | 惠州市南天生物科技有限公司 | Trichoderma spore powder as well as preparation method and application thereof |
| CN102613252A (en) * | 2011-01-27 | 2012-08-01 | 中国科学院生态环境研究中心 | Method for producing trichoderma sp. biological control agent from potato starch waste water and mushroom residues |
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| CN101874484A (en) * | 2009-12-18 | 2010-11-03 | 江苏耕耘化学有限公司 | Paste dosage-form trichoderma biopesticide composite and preparation method thereof |
| CN101874484B (en) * | 2009-12-18 | 2013-09-18 | 江苏耕耘化学有限公司 | Paste dosage-form trichoderma biopesticide composite and preparation method thereof |
| CN102067885A (en) * | 2010-03-31 | 2011-05-25 | 惠州市南天生物科技有限公司 | Trichoderma spore powder as well as preparation method and application thereof |
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| CN102613252B (en) * | 2011-01-27 | 2014-08-06 | 中国科学院生态环境研究中心 | Method for producing trichoderma sp. biological control agent from potato starch waste water and mushroom residues |
| CN103371142A (en) * | 2012-04-11 | 2013-10-30 | 中国农业科学院蔬菜花卉研究所 | Powder preparation with diatomite as carrier and application method |
| CN102676403A (en) * | 2012-05-29 | 2012-09-19 | 河北省科学院生物研究所 | Trichoderma amazonicum with induced resistance, and application thereof |
| CN102845478A (en) * | 2012-08-16 | 2013-01-02 | 上海交通大学 | Trichoderma harzianum chlamydospore granules and preparation method thereof |
| CN103918647A (en) * | 2013-01-15 | 2014-07-16 | 中国农业科学院蔬菜花卉研究所 | Biological pesticide dust agent |
| CN103141518A (en) * | 2013-03-04 | 2013-06-12 | 沈阳农业大学 | Preparation method and application of multi-effect compound anti-biological inoculants |
| CN103141519A (en) * | 2013-03-04 | 2013-06-12 | 沈阳农业大学 | Preparation method and application of anti-biological inoculants for controlling crop soil-borne diseases |
| CN103141519B (en) * | 2013-03-04 | 2015-04-01 | 沈阳农业大学 | Preparation method and application of anti-biological inoculants for controlling crop soil-borne diseases |
| CN116536239A (en) * | 2023-04-17 | 2023-08-04 | 广州市林业和园林科学研究院 | Phosphate-dissolving microbial agent, and preparation and application thereof |
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