Trichoderma harzianum solid preparation for preventing and controlling potato root rot and preparation method thereof
阅读说明：本技术 一种用于防控马铃薯根腐病的哈茨木霉固体制剂及其制备方法 (Trichoderma harzianum solid preparation for preventing and controlling potato root rot and preparation method thereof ) 是由 王喜刚 郭成瑾 沈瑞清 焦杨 张丽荣 杨波 于 2019-07-22 设计创作，主要内容包括：本发明涉及一种用于防控马铃薯根腐病的哈茨木霉固体制剂及其制备方法,其步骤为菌种活化,种子液培养,固体发酵,孢子分离,固体培养基粉碎和制剂制备,得到哈茨木霉颗粒剂或可湿性粉剂。本发明在固体发酵培养基中加入中药药渣,发酵结束后将发酵产物干燥分离,得到哈茨木霉孢子粉,再将发酵培养基干燥后粉碎,加载体与孢子粉混合制成固体制剂,该固体制剂对木贼镰刀菌、接骨木镰刀菌、尖孢镰刀菌、茄病镰刀菌、锐顶镰刀菌等马铃薯根腐病的致病菌有很好的抑制效果,可以应用于马铃薯根腐病的防治过程,从而减少和防治马铃薯根腐病的发生,提高马铃薯的产量和质量。(The invention relates to a trichoderma harzianum solid preparation for preventing and controlling potato root rot and a preparation method thereof. According to the invention, traditional Chinese medicine dregs are added into a solid fermentation culture medium, a fermentation product is dried and separated after fermentation is finished to obtain trichoderma harzianum spore powder, the fermentation culture medium is dried and crushed, and a carrier and the spore powder are added to be mixed to prepare a solid preparation.)
1. A trichoderma harzianum solid preparation for preventing and controlling potato root rot is characterized in that the active ingredients of the preparation are trichoderma harzianum spores and a solid culture medium crushing matrix containing traditional Chinese medicine dregs.
2. The trichoderma harzianum solid preparation for preventing and controlling potato root rot according to claim 1, wherein the mass ratio of the trichoderma harzianum spores to the ground substrate of the solid culture medium containing traditional Chinese medicine dregs is 1: 10-50.
3. The solid preparation of trichoderma harzianum for preventing and controlling potato root rot according to claim 1, wherein the dosage form of the trichoderma spore preparation is granules or wettable powder.
4. A preparation method of a trichoderma harzianum solid preparation for preventing and controlling potato root rot is characterized by comprising the following steps:
step one, strain activation: inoculating Trichoderma harzianum original strain M-17 strain to a PDA culture medium inclined plane, and culturing for 5-7 d at 25-28 ℃ under a dark condition;
step two, seed liquid culture: inoculating the activated strain obtained in the step one into a sterilized seed culture medium for culture;
step three, solid fermentation: inoculating the seed solution cultured in the step two into a sterilized solid fermentation culture medium, and obtaining a solid fermentation product containing trichoderma harzianum spores when hyphae overgrow the culture medium;
step four, spore separation: naturally drying the solid fermentation product in the air, controlling the water content to be 5-20%, and collecting trichoderma harzianum spores through a 100-mesh sieve;
step five, crushing a solid culture medium: further drying the collected solid culture medium of the trichoderma harzianum spores at the drying temperature of not more than 80 ℃ until the moisture content is 5-10%, crushing the dried solid culture medium, and sieving the crushed solid culture medium with a 60-mesh sieve for later use;
step six, preparation: mixing the trichoderma harzianum spores and the ground medium of the culture medium according to a proportion, and adding other carriers to prepare the trichoderma harzianum solid preparation.
5. The preparation method of the trichoderma harzianum solid preparation for preventing and controlling potato root rot according to claim 4, wherein the preparation method of the PDA culture medium in the first step comprises the following steps:
1) firstly, weighing the components of the culture medium according to the following mixture ratio: 200g of peeled potatoes, 20g of glucose, 15-20 g of agar and 1000ml of distilled water;
2) cutting potatoes into small pieces, putting the small pieces into a pot, adding 1000ml of water, heating the small pieces on a heater until the water is boiled, maintaining the boiling for 20-30 min, filtering the small pieces on a measuring cup by using 2 layers of gauze while the small pieces are hot, removing filter residues, and supplementing distilled water into filtrate to 1000 ml;
3) putting the filtrate into a pot, adding 20g of glucose and 15-20 g of agar, then putting the mixture on an asbestos net, heating the mixture with soft fire, continuously stirring the mixture by using a glass rod, subpackaging the mixture after the agar is completely dissolved, sterilizing the mixture at 121 ℃ for 25-30 min, naturally cooling the mixture, pouring the mixture into a slope and a flat plate, and solidifying the mixture for later use.
6. The preparation method of the trichoderma harzianum solid preparation for preventing and controlling potato root rot according to claim 4, wherein in the second step, the seed culture medium is 15-20 g/L of sucrose, 3-6 g/L of yeast powder and FeSO4·7H2O 0.1～0.3g/L，MgSO4·7H2O 1.2～1.5g/L，KH2PO43.5～4.5g/L，NaNO36.5-7.0 g/L; the culture medium is sterilized by moist heat at 121 deg.C25-30 min; the inoculation amount is as follows; the culture condition is that the culture is carried out for 3-6 days at 25-28 ℃ of a shaking table under the dark condition, and the rotation speed of the shaking table is 180-220 rpm.
7. The preparation method of the trichoderma harzianum solid preparation for preventing and controlling the potato root rot according to claim 4, wherein in the third step, the solid fermentation medium comprises 30-70% of traditional Chinese medicine slag powder, 10-20% of potato whole powder, 10-30% of corn flour, 0-15% of sawdust and 5-7% of potato peel, and the water addition amount is 70-80% of the mass of the components; the sterilization conditions comprise steam sterilization, the temperature is 121-125 ℃, the temperature is maintained for 45-60 min, 10-15% of seed liquid is inoculated after cooling, static culture is carried out for 3d at the temperature of 25 ℃, the culture is continued for 3-6 d after turning and stirring once, the culture process is ventilated for four times every day, the ventilation quantity is 0.2-0.5 vvm, the relative humidity is controlled to be 55-80%, and solid fermentation is finished after hyphae grow over the culture medium.
Wherein the traditional Chinese medicine residue powder is obtained by drying and crushing the residual dregs after the traditional Chinese medicine is decocted and sieving the dregs with a 60-mesh sieve, and comprises 0-100% of licorice medicine residue powder, 0-100% of isatis root dregs powder and 0-60% of wine peel dregs according to the mass.
8. The preparation method of the trichoderma harzianum solid preparation for preventing and controlling potato root rot according to claim 4, wherein the preparation steps of the granule and the wettable powder are as follows:
A. the preparation process of the trichoderma harzianum granules comprises the following steps:
mixing trichoderma harzianum spores and a solid culture medium crushed matrix containing traditional Chinese medicine dregs according to a mass ratio of 1: 100-500, adding one or more of starch slurry with a concentration of 0.1-1% w/v, sodium carboxymethyl cellulose, hydroxypropyl cellulose and methyl cellulose common binders, wherein the weight of the binder solution is 1-3% of the expected total weight of the granules, and fully and uniformly mixing; adding one or more of rice bran powder, white carbon black, bentonite, diatomite and light calcium carbonate common coating agents into the uniformly mixed solid fermentation carrier, wherein the weight of the coating agents accounts for 10-90% of the total weight of the preparation of the granules, fully and uniformly mixing, drying at the temperature of below 50 ℃, and packaging to obtain the trichoderma spore granules;
B. the preparation process of the Trichoderma harzianum wettable powder comprises the following steps:
drying the solid culture medium containing the traditional Chinese medicine dregs after collecting the spores at the temperature of below 80 ℃, crushing the solid culture medium by using a low-temperature air flow crusher, mixing the crushed solid culture medium with the trichoderma harzianum spores, adding a dispersing agent and a wetting agent, and uniformly mixing to prepare wettable powder. The dispersant is one or a combination of several of sodium dodecyl benzene sulfonate, NO, NNO, MF and sodium lignin sulfonate, and the weight of the dispersant is 2-10% of the expected total weight of the wettable powder; the wetting agent is one or a combination of more of tea seed cake, Chinese honeylocust fruit, lauryl sodium sulfate, fatty alcohol-polyoxyethylene ether and alkylphenol polyoxyethylene, and the weight of the wetting agent is 0.5-3% of the expected total weight of the wettable powder; the carrier is one or a combination of more of diatomite, white carbon black, bentonite, light calcium carbonate, attapulgite and kaolin, and the weight of the carrier is 10-90% of the expected total weight of the wettable powder.
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a trichoderma harzianum solid preparation for preventing and controlling potato root rot and a preparation method thereof.
Background
Trichoderma harzianum, a biocontrol bacterium, can be used to prevent plant diseases caused by pathogenic bacteria such as Pythium, Rhizoctonia solani, Fusarium, Rhizopus nigricans, Staphylotrichum, Sclerotinia sclerotiorum, etc. The fusarium root rot, the dry rot and the blight caused by infecting the potatoes by fusarium are world soil-borne fungal diseases, the root rot of the potatoes can be attacked in seedling culture periods and field growth periods, the fusarium root rot of the potatoes caused by various fusarium can seriously damage the production of the potatoes in recent years, the yield of commonly attacked areas is reduced by 13-35%, and the potatoes die in serious areas and even die in top of the areas. The control of the potato root rot is generally treated by adopting a chemical bactericide, the residue problem often exists when the chemical bactericide is used for treatment, simultaneously fusarium in a production field can not remove roots and is easy to relapse in the coming year, and the like, so that the trichoderma harzianum is used for biologically controlling the root rot, the control is environment-friendly, safe and effective, and the control can be carried out for a long time.
At present, more than 10 commercial trichoderma preparations are published abroad, and most trichoderma spore preparations are trichoderma preparations, but trichoderma still has many problems in plant disease control, is influenced by factors such as temperature, humidity, pH and the like in soil after being applied, has unsatisfactory colonization ability, is difficult to fully exert pesticide effect, and has insufficient remarkable growth promoting and yield increasing effects of the trichoderma on plants.
In view of the above disadvantages, various studies have been conducted at home and abroad, and patent CN201811550295.4 discloses a trichoderma harzianum solid fermentation method, a solid fermentation product, trichoderma harzianum agent, and a preparation method and application thereof, which can effectively prevent and treat grape downy mildew and grape gray mold, and can also effectively promote grape growth and improve crop yield. Patent CN200810154241.6 discloses a production process for fermenting biocontrol trichoderma by using Chinese herbal medicine dregs, wherein the biocontrol trichoderma is produced by fermenting the Chinese herbal medicine dregs in the pharmaceutical process of a Chinese pharmaceutical factory as a part of culture medium components, and the two patents do not describe how the obtained biocontrol trichoderma is active, how the biocontrol trichoderma has an effect on production, and the prevention and treatment of related plant diseases and the promotion of yield increase are not obvious.
Disclosure of Invention
Based on the prior art, the invention aims to provide a trichoderma harzianum solid preparation for preventing and controlling potato root rot and a preparation method thereof, the prepared solid preparation has good inhibition effect on pathogenic bacteria of the potato root rot such as fusarium equiseti, fusarium sambucinum, fusarium oxysporum, fusarium solani, fusarium acuminatum and the like, and can be applied to the prevention and control process of the potato root rot, so that the occurrence of the potato root rot is reduced and prevented, and the yield and the quality of potatoes are improved.
The strain Trichoderma harzianum M-17 used in the invention has a preservation number: CCTCC NO: m2018538, the Latin article name is Trichoderma harzianum M-17, the preservation unit is China center for type culture Collection, the preservation date is 8 months and 13 days in 2018, and the preservation address is Wuhan university in Wuhan, China.
The technical scheme adopted by the invention is as follows: a trichoderma harzianum solid preparation for preventing and controlling potato root rot comprises trichoderma harzianum spores and a solid culture medium crushing matrix containing traditional Chinese medicine dregs as effective components.
In order to better realize the invention, the mass ratio of the trichoderma harzianum spores to the ground substrate of the solid culture medium containing the traditional Chinese medicine dregs is 1: 10-50.
In order to better implement the invention, the trichoderma spore preparation is further prepared into granules or wettable powder.
A preparation method of a trichoderma harzianum solid preparation for preventing and controlling potato root rot comprises the following steps:
step one, strain activation: inoculating Trichoderma harzianum original strain M-17 strain to a PDA culture medium inclined plane, and culturing for 5-7 d at 25-28 ℃ under a dark condition;
step two, seed liquid culture: inoculating the activated strain obtained in the step one into a sterilized seed culture medium for culture;
step three, solid fermentation: inoculating the seed solution cultured in the step two into a sterilized solid fermentation culture medium, obtaining a solid fermentation product containing trichoderma harzianum spores after hyphae overgrow the culture medium, and counting the spores after fermentation;
step four, spore separation: naturally drying the solid fermentation product in the air, controlling the water content to be 5-20%, and collecting trichoderma harzianum spores through a 100-mesh sieve;
step five, crushing a solid culture medium: further drying the collected solid culture medium of the trichoderma harzianum spores at the drying temperature of not more than 80 ℃ until the moisture content is 5-10%, crushing the dried solid culture medium, and sieving the crushed solid culture medium with a 60-mesh sieve for later use;
step six, preparation: mixing the trichoderma harzianum spores and the ground medium of the culture medium according to a proportion, and adding other carriers to prepare the trichoderma harzianum solid preparation.
In order to better implement the invention, further, the method for preparing the PDA culture medium in the step one comprises the following steps:
1) firstly, weighing the components of the culture medium according to the following mixture ratio: 200g of peeled potatoes, 20g of glucose, 15-20 g of agar and 1000ml of distilled water;
2) cutting potatoes into small pieces, putting the small pieces into a pot, adding 1000ml of water, heating the small pieces on a heater until the water is boiled, maintaining the boiling for 20-30 min, filtering the small pieces on a measuring cup by using 2 layers of gauze while the small pieces are hot, removing filter residues, and supplementing distilled water into filtrate to 1000 ml;
3) putting the filtrate into a pot, adding 20g of glucose and 15-20 g of agar, then putting the mixture on an asbestos net, heating the mixture with soft fire, continuously stirring the mixture by using a glass rod, subpackaging the mixture after the agar is completely dissolved, sterilizing the mixture at 121 ℃ for 25-30 min, naturally cooling the mixture, pouring the mixture into a slope and a flat plate, and solidifying the mixture for later use.
In order to better realize the method, in the second step, the seed culture medium is 15-20 g/L of sucrose, 3-6 g/L of yeast powder and FeSO4·7H2O 0.1～0.3g/L，MgSO4·7H2O 1.2～1.5g/L，KH2PO43.5～4.5g/L，NaNO36.5-7.0 g/L; the moist heat sterilization condition of the culture medium is 121 ℃, and the time is 25-30 min; the inoculation amount is as follows; the culture condition is that the culture is carried out for 3-6 days at 25-28 ℃ of a shaking table under the dark condition, and the rotation speed of the shaking table is 180-220 rpm.
In order to better realize the method, further, in the third step, the solid fermentation medium comprises 30-70% of traditional Chinese medicine dreg powder, 10-20% of potato whole powder, 10-30% of corn flour, 0-15% of sawdust and 5-7% of potato peel, and the water addition amount is 70-80% of the mass of the components; sterilizing by steam, maintaining at 121-125 ℃ for 45-60 min, cooling, inoculating 10-15% seed liquid, statically culturing at 25 ℃ for 3d, continuously culturing for 3-6 d after turning and stirring once, ventilating for 30min each time in the culture process, wherein the ventilation amount is 0.2-0.5 vvm, the relative humidity is controlled to be 55-80%, after hyphae grow over the culture medium, solid fermentation is finished, counting spores after fermentation is finished, and the number of spores is 2-8 multiplied by 108One per gram.
Wherein the traditional Chinese medicine residue powder is obtained by drying and crushing the residual dregs after the traditional Chinese medicine is decocted and sieving the dregs with a 60-mesh sieve, and comprises 0-100% of licorice medicine residue powder, 0-100% of isatis root dregs powder and 0-60% of wine peel dregs according to the mass.
The spore counting and measuring method comprises the following steps: 0.5g of the solid fermentation product was diluted 100-fold with sterile water containing 0.1% v/v glycerol, stirred for 20min on a magnetic stirrer, and the number of spores was counted under a microscope using a hemocytometer.
In order to better implement the invention, the preparation steps of the six granules and the wettable powder are as follows:
A. the preparation process of the trichoderma harzianum granules comprises the following steps:
mixing trichoderma harzianum spores and a solid culture medium crushed matrix containing traditional Chinese medicine dregs according to a mass ratio of 1: 100-500, adding one or more of starch slurry with a concentration of 0.1-1% w/v, sodium carboxymethyl cellulose, hydroxypropyl cellulose and methyl cellulose common binders, wherein the weight of the binder solution is 1-3% of the expected total weight of the granules, and fully and uniformly mixing; adding one or more of rice bran powder, white carbon black, bentonite, diatomite and light calcium carbonate common coating agents into the uniformly mixed solid fermentation carrier, wherein the weight of the coating agents accounts for 10-90% of the total weight of the preparation of the granules, fully and uniformly mixing, drying at the temperature of below 50 ℃, and packaging to obtain the trichoderma spore granules;
B. the preparation process of the Trichoderma harzianum wettable powder comprises the following steps:
drying the solid culture medium containing the traditional Chinese medicine dregs after collecting the spores at the temperature of below 80 ℃, crushing the solid culture medium by using a low-temperature air flow crusher, mixing the crushed solid culture medium with the trichoderma harzianum spores, adding a dispersing agent and a wetting agent, and uniformly mixing to prepare wettable powder. The dispersant is one or a combination of several of sodium dodecyl benzene sulfonate, NO, NNO, MF and sodium lignin sulfonate, and the weight of the dispersant is 2-10% of the expected total weight of the wettable powder; the wetting agent is one or a combination of more of tea seed cake, Chinese honeylocust fruit, lauryl sodium sulfate, fatty alcohol-polyoxyethylene ether and alkylphenol polyoxyethylene, and the weight of the wetting agent is 0.5-3% of the expected total weight of the wettable powder; the carrier is one or a combination of more of diatomite, white carbon black, bentonite, light calcium carbonate, attapulgite and kaolin, and the weight of the carrier is 10-90% of the expected total weight of the wettable powder.
Advantageous effects
The invention has the following beneficial effects and innovation points:
1. when the solid preparation is prepared by fermentation, part of the Chinese medicine residues added in the solid culture medium can be used as supplement of a carbon source required by the growth of microorganisms and can reduce the production cost of the culture medium, the spore yield of trichoderma harzianum is improved after fermentation, and the antagonistic effect on fusarium is obviously improved;
2. compared with agricultural and sideline products used in other fermentation culture media, such as soybean cake powder, yeast powder, corn steep liquor, starch, dextrin and the like, the research adds the Chinese medicine residues to prepare the trichoderma harzianum fermentation preparation, is one of innovations of the patent, changes waste into valuable, reduces the environmental pollution of the Chinese medicine residues, and has the advantages of unique formula, environmental protection, safety and high efficiency;
3. the prepared solid preparation, whether granules or wettable powder, uses solid medium residues as a carrier, recycles waste residues, reduces environmental pollution, and improves the control effect of the product by using beneficial substances after the fermentation of the Chinese medicine residues;
4. the preparation prepared by the invention is a live spore preparation, the spore treatment temperature is not more than 50 ℃ in the production process, the use is convenient when the preparation is used for preventing and treating the potato root rot, and the effect is obvious;
5. the preparation method of the granules and the wettable powder is used for trichoderma harzianum for the first time, and an ideal effect is achieved.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
The preparation method of the PDA culture medium comprises the following steps:
1) firstly, weighing the components of the culture medium according to the following mixture ratio: 200g of peeled potatoes, 20g of glucose, 15-20 g of agar and 1000ml of distilled water;
2) cutting potatoes into small pieces, putting the small pieces into a pot, adding 1000ml of water, heating the small pieces on a heater until the water is boiled, maintaining the boiling for 20-30 min, filtering the small pieces on a measuring cup by using 2 layers of gauze while the small pieces are hot, removing filter residues, and supplementing distilled water into filtrate to 1000 ml;
3) putting the filtrate into a pot, adding 20g of glucose and 15-20 g of agar, then putting the mixture on an asbestos net, heating the mixture with soft fire, continuously stirring the mixture by using a glass rod, subpackaging the mixture after the agar is completely dissolved, sterilizing the mixture at 121 ℃ for 25-30 min, naturally cooling the mixture, pouring the mixture into a slope and a flat plate, and solidifying the mixture for later use.