CN110699259A - Trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot and preparation method of preparation - Google Patents
Trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot and preparation method of preparation Download PDFInfo
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Abstract
The invention relates to a trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot and a preparation method of a preparation. The liquid preparation or the solid preparation has good inhibition effect on pathogenic bacteria of the potato root rot such as fusarium equiseti, fusarium sambuci, fusarium oxysporum, fusarium solani, fusarium acuminatum and the like, and can be applied to the control process of the potato root rot, thereby reducing and controlling the occurrence of the potato root rot and improving the yield and the quality of the potatoes.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot and a preparation method of a preparation.
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, the two patents do not describe how the obtained biocontrol trichoderma is active and the problem of yield in the trichoderma fermentation process is solved, and the effects on production are not obvious in prevention and treatment of related plant diseases and promotion of yield increase.
Disclosure of Invention
Based on the prior art, the invention aims at providing a trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot and a preparation method of a preparation, the method can obviously improve the yield of trichoderma harzianum through liquid fermentation, and the prepared preparation has good inhibition effect on pathogenic bacteria of the potato root rot, such as fusarium equiseti, fusarium sambuci, 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 liquid fermentation method for preventing and controlling potato root rot comprises the following steps:
step one, slant culture, inoculating Trichoderma harzianum original strain M-17 strain to PDA culture medium slant for culture;
step two, seed liquid culture, namely inoculating the slant strains cultured in the step one into a sterilized seed culture medium for culture;
step three, liquid fermentation, namely inoculating the seed liquid cultured in the step two into a sterilized liquid fermentation culture medium for culture to obtain a trichoderma harzianum fermented product;
step four, preparation, namely preparing the liquid fermentation liquor which is fermented in the step three into a liquid preparation or a 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 small pieces are boiled, maintaining the boiling for 20-30 min, and filtering the small pieces on a measuring cup, removing filter residues, and supplementing distilled water to the filtrate to 1000ml according to the method for fermenting trichoderma harzianum for preventing and controlling potato root rot according to claim 1 when 2 layers of gauze are hot;
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 invention, the first step is that the slant culture condition is dark condition, and the culture is carried out for 5-7 days at 25-28 ℃;
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, 0.01-0.05 g/L of NaCl 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 1-2 cm2Slant spores; 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 invention, the liquid fermentation medium in the third step is one or more of wheat flour, corn flour, potato starch, soluble starch and bran powder with the carbon source addition amount of 2%; the nitrogen source is added with 2 percent of one or more of soybean meal, grape wine peel residue powder, ammonium nitrate, urea, ammonium sulfate and sodium nitrate; the addition amount of other microelements is 1.4 percent of one or more of magnesium sulfate, calcium nitrate and ferrous sulfate; the growth promoter is one or more of leachate of Glycyrrhrizae radix and radix Isatidis, and the concentration is 30%, 50%, and 70%, respectively.
In order to better realize the method, in the third step, the sterilization parameter of the liquid fermentation medium is steam moist heat sterilization, the temperature is maintained at 121-124 ℃ for 25-35 min, the culture condition is culture at 25-28 ℃, the ventilation ratio is 1:0.7v/v, stirring is started at 90-115 rpm, and the culture lasts for 10-13 d.
In order to better implement the invention, further, the preparation method of the preparation in the step four comprises the following steps:
the preparation process of the Trichoderma harzianum liquid preparation comprises the following steps:
after the fermentation of the trichoderma harzianum liquid is finished, the concentration of the fermentation liquid is more than 50%, the pH value is 4.5-5.5, sterilized 0.1-0.5 per mill of glycerol and 0.05-0.1 per mill of vegetable oil are added, and the sterilization condition is 45-60 min at 125 ℃; mixing, stirring, and aseptically packaging to obtain liquid preparation;
the bacterial concentration detection method comprises the following steps: centrifuging 10g of fermentation liquor, centrifuging at 3000rpm for 10min, removing supernatant, dividing the remainder by 10g of fermentation liquor, and taking percentage to obtain the bacterial concentration which is relative thallus concentration.
The preparation process of the Trichoderma harzianum solid preparation comprises the following steps:
after the fermentation of the trichoderma harzianum liquid is finished, the concentration of the fermentation liquid is more than 50%, the pH value is 4.5-5.5, plate-and-frame filtration is carried out, the filtered mycelium is dried at 35-45 ℃ to prepare solid granules, one or a combination of more of starch slurry with the concentration of 0.1-1% w/v, sodium carboxymethyl cellulose, hydroxypropyl cellulose and methyl cellulose common binders are added, the weight of the binder solution is 1-3% of the expected total weight of the granules, and the mixture is fully and uniformly mixed; and adding one or more of traditional Chinese medicine powder, rice bran powder, white carbon black, bentonite, diatomite and light calcium carbonate into the uniformly mixed solid fermentation carrier, wherein the traditional Chinese medicine powder is a dried and crushed product of residual traditional Chinese medicine residues after the traditional Chinese medicine leachate is used in liquid fermentation, the weight of the coating agent accounts for 10-90% of the total weight of the expected preparation of the granules, fully and uniformly mixing, drying at the temperature of below 50 ℃ and packaging to obtain the trichoderma harzianum granules.
In order to better realize the invention, further, the liquid fermentation is one of corn flour and bran flour with the carbon source addition amount of 2%; the nitrogen source is added into 2 percent of one of soybean meal and wine peel residue powder; the addition amount of the trace elements is 1.4 percent of one or more of magnesium sulfate, calcium nitrate and ferrous sulfate; the growth promoter is one or more of leachate of Glycyrrhrizae radix and radix Isatidis, and the concentration is 30%, 50%, and 70%, respectively.
In order to better realize the invention, the preparation method of the licorice and isatis root leachate comprises the following steps: weighing liquorice and isatis root, adding three times of water by weight, decocting, boiling for 25-40 min, and filtering to obtain filtrate, namely the traditional Chinese medicine leachate.
Advantageous effects
The invention has the following beneficial effects:
1. the traditional Chinese medicine extract is used as one of the raw materials of the culture medium in the liquid fermentation, so that the yield of trichoderma harzianum spores and the resistance of the trichoderma harzianum spores to fusarium are improved;
2. the prepared liquid preparation has simple and reliable process, the viable bacteria and the products thereof are retained to the maximum extent, and the effect is better when in use;
3. the prepared solid preparation uses the traditional Chinese medicine residues as a carrier, makes up the loss in the mycelium drying process, enriches the preparation types of the liquid fermentation trichoderma harzianum, and increases the product adaptability; the prepared granules are convenient to use and good in effect, are not influenced by factors such as temperature, humidity and pH in soil after being applied, can fully exert the drug effect, can be stably produced, and are simple in production condition, low in cost and easy to realize;
4. the prepared solid preparation uses the traditional Chinese medicine residues as a carrier, changes waste into valuable, solves the problem of recycling the traditional Chinese medicine residues, reduces the environmental pollution of the traditional Chinese medicine residues, and has unique formula, environmental protection, safety and high efficiency; meanwhile, the method is beneficial to accelerating the popularization and application of trichoderma harzianum as the biocontrol treatment of the potato root rot and the sustainable development of agriculture.
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.
Example 1
The embodiment provides a trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot and a preparation method of a preparation thereof, and the method comprises the following steps:
step one, slant culture, inoculating Trichoderma harzianum original strain M-17 strain to PDA culture medium slant, and culturing at 25 ℃ for 7d under dark condition;
step two, seed liquid culture: inoculating the activated strain obtained in the step one into a sterilized seed culture medium for culture, wherein the seed culture medium comprises 15g/L of sucrose, 3g/L of yeast powder, 0.01g/L of NaCl and FeSO4·7H2O 0.1g/L，MgSO4·7H2O 1.2g/L， KH2PO43.5g/L，NaNO36.5 g/L; the culture medium is sterilized by moist heat at 121 deg.C for 25 min; the inoculation amount is 1cm2Slant spores; culturing at 25 deg.C for 6d in dark condition with shaking table rotation speed of 180 rpm;
step three, liquid fermentation: inoculating the seed solution cultured in the step two into a sterilized liquid fermentation culture medium for culturing to obtain a trichoderma harzianum fermented product; the liquid fermentation medium comprises the following components: corn flour with the carbon source addition amount of 2%, soybean flour with the nitrogen source addition amount of 2%, magnesium sulfate with the trace element addition amount of 0.5% and calcium nitrate with the trace element addition amount of 0.9%, and a growth promoter which is liquorice leachate with the concentration of 30%; the sterilization condition is steam damp heat sterilization, and the temperature is maintained at 121-124 ℃ for 25-35 min; inoculating 10% of seed liquid; culturing at 25 deg.C under the condition of ventilation ratio of 1:0.7v/v, stirring at 90rpm, and culturing for 13d to obtain Trichoderma harzianum fermentation liquid;
the preparation method of the licorice and isatis root leachate comprises the following steps: weighing liquorice and isatis root, adding three times of water by weight, decocting, boiling for 25-40 min, and filtering to obtain a filtrate, namely a traditional Chinese medicine leaching solution;
step four, preparation, namely preparing the liquid fermentation liquor fermented in the step three into a liquid preparation, wherein the preparation process comprises the following steps:
after the fermentation of the trichoderma harzianum liquid is finished, the concentration of the fermentation liquid is more than 50%, the pH value is 4.5-5.5, sterilized 0.2 thousandth of glycerin and 0.1 thousandth of vegetable oil are added, and the sterilization condition is 125 ℃ for 60 min; mixing, stirring, and aseptically packaging to obtain liquid preparation;
the bacterial concentration detection method comprises the following steps: centrifuging 10g of fermentation liquor, centrifuging at 3000rpm for 10min, removing supernatant, dividing the remainder by 10g of fermentation liquor, and taking percentage to obtain the bacterial concentration which is relative thallus concentration.
Example 2
The embodiment provides a trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot and a preparation method of a preparation thereof, and the method comprises the following steps:
step one, slant culture, inoculating Trichoderma harzianum original strain M-17 strain to a PDA culture medium slant, and culturing for 6d at 2 ℃ 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, wherein the seed culture medium comprises 16g/L of sucrose, 4g/L of yeast powder, 0.02g/L of NaCl and FeSO4·7H2O 0.2g/L，MgSO4·7H2O 1.3g/L， KH2PO43.8g/L，NaNO36.6 g/L; the culture medium is sterilized by moist heat at 121 deg.C for 28 min; the inoculation amount is 1cm2Slant spores; culturing at 25 deg.C for 5d in dark condition with shaking table rotation speed of 190 rpm;
step three, liquid fermentation: inoculating the seed solution cultured in the step two into a sterilized liquid fermentation culture medium for culturing to obtain a trichoderma harzianum fermented product; the liquid fermentation medium comprises the following components: the carbon source adding amount is 2% of bran powder, the nitrogen source adding amount is 2% of grape wine peel residue powder, and the trace elements adding amount is 0.5% of magnesium sulfate and 0.15% of ferrous sulfate; the growth promoter is 50% radix Isatidis leachate; culturing at 25 deg.C under the condition of aeration ratio of 1:0.7v/v, stirring at 115rpm, and culturing for 12d to obtain Trichoderma harzianum fermentation liquid;
the preparation method of the licorice and isatis root leachate comprises the following steps: weighing liquorice and isatis root, adding three times of water by weight, decocting, boiling for 25-40 min, and filtering to obtain a filtrate, namely a traditional Chinese medicine leaching solution;
step four, preparing the liquid fermentation liquor fermented in the step three into a liquid preparation, wherein the preparation process comprises the following steps: after the fermentation of the trichoderma harzianum liquid is finished, the concentration of the fermentation liquid is more than 50%, the pH value is 4.5-5.5, sterilized 0.5 thousandth of glycerin and 0.1 thousandth of vegetable oil are added, and the sterilization condition is 125 ℃ for 45 min; mixing, stirring, and aseptically packaging to obtain liquid preparation;
the bacterial concentration detection method comprises the following steps: centrifuging 10g of fermentation liquor, centrifuging at 3000rpm for 10min, removing supernatant, dividing the remainder by 10g of fermentation liquor, and taking percentage to obtain the bacterial concentration which is relative thallus concentration.
Example 3
The embodiment provides a trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot and a preparation method of a preparation thereof, and the method comprises the following steps:
step one, slant culture, inoculating Trichoderma harzianum original strain M-17 strain to PDA culture medium slant, and culturing at 27 deg.C for 6d under dark condition;
step two, seed liquid culture:inoculating the activated strain obtained in the step one into a sterilized seed culture medium for culture, wherein the seed culture medium comprises 20g/L of sucrose, 4g/L of yeast powder, 0.03g/L of NaCl and FeSO4·7H2O 0.2g/L，MgSO4·7H2O 1.4g/L， KH2PO44.0g/L，NaNO36.7 g/L; the culture medium is sterilized by moist heat at 121 deg.C for 30 min; the inoculation amount is 1.5cm2Slant spores; culturing at 27 deg.C for 4d in dark condition with shaking table rotation speed of 200 rpm;
step three, liquid fermentation: inoculating the seed solution cultured in the step two into a sterilized liquid fermentation culture medium for culturing to obtain a trichoderma harzianum fermented product; the liquid fermentation medium comprises the following components: corn flour with the carbon source addition amount of 2%, soybean flour with the nitrogen source addition amount of 2%, calcium nitrate with the trace element addition amount of 0.9% and ferrous sulfate with the trace element addition amount of 0.15%; the growth promoter is 70% radix Isatidis leachate; culturing at 26 deg.C under the condition of aeration ratio of 1:0.7v/v, stirring at 100rpm, and culturing for 12d to obtain Trichoderma harzianum fermentation liquid;
the preparation method of the licorice and isatis root leachate comprises the following steps: weighing liquorice and isatis root, adding three times of water by weight, decocting, boiling for 25-40 min, and filtering to obtain a filtrate, namely a traditional Chinese medicine leaching solution;
step four, preparing the liquid fermentation liquor fermented in the step three into a solid preparation, wherein the preparation process comprises the following steps:
after the fermentation of the trichoderma harzianum liquid is finished, the concentration of the fermentation liquid is more than 50%, the pH value is 4.5-5.5, plate-and-frame filtration is carried out, the filtered mycelium is air-dried at 35-45 ℃ to prepare solid granules, sodium carboxymethyl cellulose with the concentration of 0.3% w/v and a common binder are added, the weight of the binder solution is 1% of the expected total weight of the granules, and the mixture is fully and uniformly mixed; adding Chinese medicinal powder and testa oryzae powder into the uniformly mixed solid fermentation carrier, wherein the Chinese medicinal powder is obtained by fermenting liquid with the residue of Chinese medicinal extract, drying and pulverizing, the coating agent accounts for 10% of the total weight of the granule, mixing uniformly, oven drying below 50 deg.C, and packaging to obtain Trichoderma harzianum granule.
Example 4
The embodiment provides a trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot and a preparation method of a preparation thereof, and the method comprises the following steps:
step one, slant culture, inoculating Trichoderma harzianum original strain M-17 strain to PDA culture medium slant, and culturing at 28 ℃ for 5d under dark condition;
step two, seed liquid culture: inoculating the activated strain obtained in the step one into a sterilized seed culture medium for culture, wherein the seed culture medium comprises 18g/L of cane sugar, 5g/L of yeast powder, 0.04g/L of NaCl and FeSO4·7H2O 0.3g/L，MgSO4·7H2O 1.4g/L， KH2PO44.2g/L，NaNO36.8 g/L; the culture medium is sterilized by moist heat at 121 deg.C for 25 min; the inoculation amount is 1.5cm2Slant spores; culturing at 28 deg.C for 3d in dark condition with shaking table rotation speed of 200 rpm;
step three, liquid fermentation: inoculating the seed solution cultured in the step two into a sterilized liquid fermentation culture medium for culturing to obtain a trichoderma harzianum fermented product; the liquid fermentation medium comprises the following components: the carbon source adding amount is 2% of bran powder, the nitrogen source adding amount is 2% of grape wine peel residue powder, and the trace element adding amount is 1.4% of magnesium sulfate; the growth promoter is 50% Glycyrrhrizae radix extract; culturing at 28 deg.C under the condition of ventilation ratio of 1:0.7v/v, stirring at 90rpm, and culturing for 11d to obtain Trichoderma harzianum fermentation liquid;
the preparation method of the licorice and isatis root leachate comprises the following steps: weighing liquorice and isatis root, adding three times of water by weight, decocting, boiling for 25-40 min, and filtering to obtain a filtrate, namely a traditional Chinese medicine leaching solution;
step four, preparing the liquid fermentation liquor fermented in the step three into a solid preparation, wherein the preparation process comprises the following steps:
after the fermentation of the trichoderma harzianum liquid is finished, the concentration of the fermentation liquid is more than 50%, the pH value is 4.5-5.5, plate-and-frame filtration is carried out, the filtered mycelium is air-dried at 35-45 ℃ to prepare solid granules, 0.1% w/v starch slurry and 0.2% w/v sodium carboxymethyl cellulose are added, the weight of the binder solution is 2% of the expected total weight of the granules, and the components are fully and uniformly mixed; and adding one or more of traditional Chinese medicine powder, rice bran powder, white carbon black, bentonite, diatomite and light calcium carbonate into the uniformly mixed solid fermentation carrier, wherein the traditional Chinese medicine powder is a dried and crushed product of residual traditional Chinese medicine residues after the traditional Chinese medicine leachate is used in liquid fermentation, the weight of the coating agent accounts for 30% of the total weight of the expected preparation of the granules, fully and uniformly mixing, drying below 50 ℃ and packaging to obtain the trichoderma harzianum granules.
Example 5
The embodiment provides a trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot and a preparation method of a preparation thereof, and the method comprises the following steps:
step one, slant culture, inoculating Trichoderma harzianum original strain M-17 strain to PDA culture medium slant, and culturing at 25 deg.C for 5d under dark condition;
step two, seed liquid culture: inoculating the activated strain obtained in the step one into a sterilized seed culture medium for culture, wherein the seed culture medium comprises 20g/L of sucrose, 6g/L of yeast powder, 0.05g/L of NaCl and FeSO4·7H2O0.3g/L，MgSO4·7H2O1.5g/L， KH2PO44.5g/L，NaNO37.0 g/L; the culture medium is sterilized by moist heat at 121 deg.C for 30 min; the inoculation amount is 2cm2Slant spores; culturing at 25 deg.C for 6d in dark condition with shaking table rotation speed of 220 rpm;
step three, liquid fermentation: inoculating the seed solution cultured in the step two into a sterilized liquid fermentation culture medium for culturing to obtain a trichoderma harzianum fermented product; the liquid fermentation medium comprises the following components: the carbon source adding amount is 2% of bran powder, the nitrogen source adding amount is 2% of grape wine peel slag powder, the trace elements adding amount is 0.5% of magnesium sulfate, 0.8% of calcium nitrate and 0.15% of ferrous sulfate; the growth promoter is 30% of radix Glycyrrhizae extract and 50% of radix Isatidis extract; culturing at 28 deg.C under the condition of aeration ratio of 1:0.7v/v, stirring at 115rpm, and culturing for 10d to obtain Trichoderma harzianum fermentation liquid;
the preparation method of the licorice and isatis root leachate comprises the following steps: weighing liquorice and isatis root, adding three times of water by weight, decocting, boiling for 25-40 min, and filtering to obtain a filtrate, namely a traditional Chinese medicine leaching solution;
step four, preparation, namely preparing the liquid fermentation liquor fermented in the step three into a solid preparation, wherein the preparation process comprises the following steps: after the fermentation of the trichoderma harzianum liquid is finished, the concentration of the fermentation liquid is more than 50%, the pH value is 4.5-5.5, plate-and-frame filtration is carried out, the filtered mycelium is air-dried at 35-45 ℃ to prepare solid granules, 0.2% w/v starch slurry and 0.1% w/v sodium carboxymethyl cellulose are added, the weight of the binder solution is 2% of the expected total weight of the granules, and the components are fully and uniformly mixed; and adding one or more of traditional Chinese medicine powder, rice bran powder, white carbon black, bentonite, diatomite and light calcium carbonate into the uniformly mixed solid fermentation carrier, wherein the traditional Chinese medicine powder is a dried and crushed product of residual traditional Chinese medicine residues after the traditional Chinese medicine leachate is used in liquid fermentation, the weight of the coating agent accounts for 50% of the total weight of the expected preparation of the granules, fully and uniformly mixing, drying below 50 ℃ and packaging to obtain the trichoderma harzianum granules.
And adding one or more of traditional Chinese medicine powder, rice bran powder, white carbon black, bentonite, diatomite and light calcium carbonate into the uniformly mixed solid fermentation carrier, wherein the traditional Chinese medicine powder is a dried and crushed product of residual traditional Chinese medicine residues after the traditional Chinese medicine leachate is used in liquid fermentation, the weight of the coating agent accounts for 10-90% of the total weight of the expected preparation of the granules, fully and uniformly mixing, drying at the temperature of below 50 ℃ and packaging to obtain the trichoderma harzianum granules.
The liquid preparation and solid preparation granules prepared from the trichoderma harzianum prepared in the above examples 1 to 5 have the effective viable count of more than or equal to 1 hundred million/gram, and are applied to fields, and the using method is that 1kg of microbial inoculum is added with 7kg of water and mixed uniformly for seed dressing; the uniform mixture of 1kg of microbial inoculum and 100kg of soil is applied to each mu of field.
And (4) investigating the disease progression of each processed potato block dry rot during harvesting, and calculating the disease index and the prevention effect.
Grading standard of dry rot potato: level 0: no disease spots are formed on the potato blocks; level 1: the lesion spots are small, and the whole area of the potato block is less than 5 percent; and 3, level: the disease spots are small, and the area of the disease part accounts for 5-10% of the whole potato block area; and 5, stage: the disease spots are smaller or respectively larger, and the area of the disease part accounts for 11-25% of the whole area of the potato block; and 7, stage: the sizes of the disease spots are distributed, and the area of the disease spots accounts for 26-50% of the whole area of the potato block; and 9, stage: the sizes of the disease spots are distributed, and the area of the connected disease parts accounts for more than 50% of the whole area of the potato block.
Disease index ═ Σ (number of diseased potatoes at each level x relative level) x 100/(survey total number of potatoes x 9)
Relative control (%) (control disease index-treatment disease index)/control disease index) x 100%
The test results are shown in table 3:
TABLE 1 results of the determination of the control of Fusarium solani and the yield of each example
As can be seen from Table 1, the Trichoderma harzianum microbial agent obtained by the invention can reduce the incidence rate of the fusarium root rot of potatoes to different degrees, has a certain control effect on the fusarium root rot of potatoes, and can increase the yield of potatoes and improve the commodity potato rate.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the invention as defined by the appended claims. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A trichoderma harzianum liquid fermentation method for preventing and controlling potato root rot is characterized by comprising the following steps:
step one, slant culture, inoculating Trichoderma harzianum original strain M-17 strain to PDA culture medium slant for culture;
step two, seed liquid culture, namely inoculating the slant strains cultured in the step one into a sterilized seed culture medium for culture;
step three, liquid fermentation, namely inoculating the seed liquid cultured in the step two into a sterilized liquid fermentation culture medium for culture to obtain a trichoderma harzianum fermented product;
step four, preparation, namely preparing the liquid fermentation liquor which is fermented in the step three into a liquid preparation or a solid preparation.
2. The liquid fermentation method of trichoderma harzianum for preventing and controlling potato root rot according to claim 1, wherein the method for preparing the PDA medium in the first step is as follows:
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 small pieces are boiled, maintaining the boiling for 20-30 min, and filtering the small pieces on a measuring cup, removing filter residues, and supplementing distilled water to the filtrate to 1000ml according to the method for fermenting trichoderma harzianum for preventing and controlling potato root rot according to claim 1 when 2 layers of gauze are hot;
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.
3. The liquid fermentation method of trichoderma harzianum for preventing and controlling potato root rot according to claim 1, wherein the slant culture condition in the first step is a dark condition, and the culture is carried out at 25-28 ℃ for 5-7 days.
4. The liquid fermentation method of trichoderma harzianum for preventing and controlling potato root rot according to claim 1, wherein the seeds in step two areThe seed culture medium is 15-20 g/L of sucrose, 3-6 g/L of yeast powder, 0.01-0.05 g/L of NaCl 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 1-2 cm2Slant spores; 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.
5. The liquid fermentation method of trichoderma harzianum for preventing and controlling potato root rot according to claim 1, characterized in that the liquid fermentation medium in step three is one or more of wheat flour, corn flour, potato whole flour, potato starch, soluble starch and bran flour with a carbon source addition amount of 2%; the nitrogen source is added with 2 percent of one or more of soybean meal, grape wine peel residue powder, ammonium nitrate, urea, ammonium sulfate and sodium nitrate; the addition amount of other microelements is 1.4 percent of one or more of magnesium sulfate, calcium nitrate and ferrous sulfate; the growth promoter is one or more of leachate of Glycyrrhrizae radix and radix Isatidis, and the concentration is 30%, 50%, and 70%, respectively.
6. The liquid fermentation method of trichoderma harzianum for preventing and controlling potato root rot according to claim 1, characterized in that in the third step, the sterilization parameters of the liquid fermentation medium are steam moist heat sterilization, the temperature is maintained at 121-124 ℃ for 25-35 min, the culture conditions are culture at 25-28 ℃, the ventilation ratio is 1:0.7v/v, the stirring is started at 90-115 rpm, and the culture lasts for 10-13 d.
7. The liquid fermentation method of trichoderma harzianum for preventing and controlling potato root rot according to claim 1, wherein the preparation method in the fourth step is:
the preparation process of the Trichoderma harzianum liquid preparation comprises the following steps:
after the fermentation of the trichoderma harzianum liquid is finished, the concentration of the fermentation liquid is more than 50%, the pH value is 4.5-5.5, sterilized 0.1-0.5 per mill of glycerol and 0.05-0.1 per mill of vegetable oil are added, and the sterilization condition is 45-60 min at 125 ℃; mixing, stirring, and aseptically packaging to obtain liquid preparation;
the bacterial concentration detection method comprises the following steps: centrifuging 10g of fermentation liquor, centrifuging at 3000rpm for 10min, removing supernatant, dividing the remainder by 10g of fermentation liquor, and taking percentage to obtain the bacterial concentration which is relative thallus concentration.
The preparation process of the Trichoderma harzianum solid preparation comprises the following steps:
after the fermentation of the trichoderma harzianum liquid is finished, the concentration of the fermentation liquid is more than 50%, the pH value is 4.5-5.5, plate-and-frame filtration is carried out, the filtered mycelium is dried at 35-45 ℃ to prepare solid granules, one or a combination of more of starch slurry with the concentration of 0.1-1% w/v, sodium carboxymethyl cellulose, hydroxypropyl cellulose and methyl cellulose common binders are added, the weight of the binder solution is 1-3% of the expected total weight of the granules, and the mixture is fully and uniformly mixed; and adding one or more of traditional Chinese medicine powder, rice bran powder, white carbon black, bentonite, diatomite and light calcium carbonate into the uniformly mixed solid fermentation carrier, wherein the traditional Chinese medicine powder is a dried and crushed product of residual traditional Chinese medicine residues after the traditional Chinese medicine leachate is used in liquid fermentation, the weight of the coating agent accounts for 10-90% of the total weight of the expected preparation of the granules, fully and uniformly mixing, drying at the temperature of below 50 ℃ and packaging to obtain the trichoderma harzianum granules.
8. The liquid fermentation method of trichoderma harzianum for preventing and controlling potato root rot according to claim 4, characterized in that the liquid fermentation is one of corn flour and bran flour with 2% of carbon source addition; the nitrogen source is added into 2 percent of one of soybean meal and wine peel residue powder; the addition amount of the trace elements is 1.4 percent of one or more of magnesium sulfate, calcium nitrate and ferrous sulfate; the growth promoter is one or more of leachate of Glycyrrhrizae radix and radix Isatidis, and the concentration is 30%, 50%, and 70%, respectively.
9. The liquid fermentation method of trichoderma harzianum for preventing and controlling potato root rot according to claim 5, wherein the licorice and isatis root leachate is prepared by the following steps: weighing liquorice and isatis root, adding three times of water by weight, decocting, boiling for 25-40 min, and filtering to obtain filtrate, namely the traditional Chinese medicine leachate.
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