CN105802863B - Trichoderma harzianum fermentation process, wettable powder and application thereof - Google Patents
Trichoderma harzianum fermentation process, wettable powder and application thereof Download PDFInfo
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Abstract
The invention discloses a Trichoderma harzianum fermentation process, wettable powder and application thereof, and relates to the fields of fermentation processes, wettable powder and application thereof. The invention aims to solve the problems of high cost and poor plant disease control effect of stable wettable powder prepared from common Trichoderma harzianum strains. The fermentation process of trichoderma harzianum is carried out according to the following steps: firstly, selecting and processing raw materials; secondly, preparing a trichoderma harzianum solid state fermentation culture medium; and thirdly, fermenting trichoderma harzianum. The preparation method of the wettable powder comprises the following steps: evenly mixing conidium powder obtained by fermenting trichoderma harzianum, a wetting agent, a dispersing agent, a synergist X and a carrier to obtain wettable powder. The wettable powder has the function of promoting germination of tomato seeds, and has obvious control effects on tomato powdery mildew, gray mold and leaf mold. The Trichoderma harzianum fermentation process and the preparation of the wettable powder are used for promoting tomato seed germination and preventing and treating tomato diseases.
Description
Technical Field
The invention relates to a fermentation process, wettable powder and the application field thereof.
Background
The tomato is one of the most widely cultivated and applied vegetables in China, and the tomato industry in China has a good development situation in recent years. However, in production, diseases are always a great problem to producers. In the vast production line, especially in the out-of-season cultivation of protected areas, a plurality of diseases often occur simultaneously. At present, the control of tomato diseases in the production process is mainly chemical control. Chemical pesticides are used in large quantities for a long time, so that the environment is seriously polluted, and the pesticide residues can also damage the health of people and livestock; moreover, pathogenic bacteria are easy to generate drug resistance, so that the control effect is reduced year by year. Therefore, the biopesticides and biological control technologies which are environment-friendly, safe and effective are gradually receiving attention from people.
Biological control is to control plant diseases and insect pests by using the relationship between natural species. The biocontrol fungus is an important member of biological pesticides, is a natural object in the nature, and cannot pollute the environment and destroy the ecological balance. It has various disease prevention mechanisms and can effectively reduce the drug resistance of pathogenic bacteria. Most antagonistic bacteria also stimulate the growth and development of plants. The plant resistance can be induced, the self defense system of the plant can be activated, the plant can resist the invasion of pathogenic bacteria, the growth and development of the plant can be promoted, and the effects of promoting yield and increasing income can be achieved.
Trichoderma harzianum is a biocontrol bacterium, has good adaptability, wide growth range and various disease prevention mechanisms, can act on pathogenic bacteria through competitive action, heavy parasitic action, secretion of antibiotics and other ways, and has obvious control effect on a plurality of plant diseases through a plurality of researches. But the commercial preparations in domestic market are few, and the price of the existing preparation is higher, which is determined by the production cost; the external environment has a large influence on the microbial inoculum, and the prevention and treatment effect is unstable. Therefore, the key point of the popularization of the biopesticides is to grasp the principle of 'cost reduction and stable effect'.
Disclosure of Invention
The invention aims to solve the problems of high cost and poor plant disease control effect of stable wettable powder prepared from common trichoderma harzianum strains, and provides a trichoderma harzianum fermentation process, wettable powder and application.
The fermentation process of trichoderma harzianum is carried out according to the following steps:
firstly, selecting and processing raw materials: selecting domestic garbage or agricultural wastes as raw materials, drying and then crushing for later use by a crusher; the domestic garbage comprises folium sarcandrae, grape leaves and pineapple peels, and the agricultural wastes comprise corncobs and wheat bran;
secondly, preparing a trichoderma harzianum solid state fermentation culture medium: adding the sardine leaves, the grape leaves, the corncobs, the pineapple peels and the wheat bran into a container to obtain a trichoderma harzianum solid state fermentation culture medium; the mass ratio of the Chinese hawthorn leaf to the grape leaf to the corn cob to the pineapple peel to the wheat bran is (3-6): 1-2): 20-41;
and thirdly, fermentation of trichoderma harzianum: and (3) adding the Trichoderma harzianum WY-1 bacterial liquid into the Trichoderma harzianum solid fermentation culture medium prepared in the step two, wherein the culture conditions are as follows: the initial water content is 80-100%, the inoculation amount is 6-10%, and the fermentation of trichoderma harzianum is completed after 10d-12d of culture at 25-28 ℃ to obtain conidium powder; the preparation method of the trichoderma harzianum liquid comprises the following steps: picking and inoculating Trichoderma harzianum WY-1 into potato glucose culture medium, and carrying out shake flask culture at 25-28 ℃ for 2-4 days, wherein the Trichoderma harzianum WY-1 is preserved in China general microbiological culture Collection center, addresses: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is 2016, 4 and 11 months, and the preservation number is CGMCC No. 12165.
The wettable powder prepared from the conidium powder obtained by the trichoderma harzianum fermentation process comprises 1-2% of the conidium powder obtained by trichoderma harzianum fermentation, 5-8% of a wetting agent, 3-6% of a dispersing agent and the balance of a carrier according to the mass fraction; the wetting agent is one or a composition of more of sodium dodecyl benzene sulfonate, nekal, fatty alcohol-polyoxyethylene ether and tween-80; the dispersing agent is one or a composition of more of sodium lignosulphonate, 2-naphthalene sulfonic acid formaldehyde polymer sodium salt, carboxymethyl cellulose and polyethylene glycol 10000; the carrier is one or a combination of more of kaolin, diatomite, talcum powder, Ca-based bentonite, attapulgite and zeolite powder.
The preparation method of the wettable powder is carried out according to the following steps: evenly mixing conidium powder obtained by fermenting trichoderma harzianum, a wetting agent, a dispersing agent, a synergist X and a carrier to obtain wettable powder.
The application of the wettable powder of the invention is as follows: the tomato seed germination promoter has the effect of promoting germination of tomato seeds, and has remarkable control effects on tomato powdery mildew, gray mold and leaf mold; specifically, 100-10000 times of liquid of Trichoderma harzianum wettable powder has obvious effect of promoting germination of tomato seeds, and 1000-10000 times of liquid of Trichoderma harzianum wettable powder has obvious effect of preventing and treating powdery mildew, gray mold and leaf mold of tomato.
Compared with the prior art, the invention has the advantages that:
1. in the preparation of the powder, the condition required by enabling trichoderma harzianum to better adapt to the environment and quickly colonize is considered, the created synergist is matched with the trichoderma harzianum powder for use, and the powdery mildew is prevented and treated, so that the treatment effect is good, and the pesticide effect duration is long. The 1000-time liquid of the Trichoderma harzianum wettable powder has obvious control effects on tomato powdery mildew, gray mold and leaf mold, the control effects respectively reach 82.2%, 56.8% and 72.4%, and the control effects are superior to those of commercial Trichoderma harzianum powder (57.8%, 41.9% and 55.8) and chemical pesticides (61.6%, 47.2% and 43.6%). The powder has obvious germination promotion effect on tomato seeds, and the highest germination potential of the tomato seeds treated by soaking the seeds in 1000 times of 200-fold liquid is 2-3 days earlier than that of the untreated tomato seeds. The 200-time solution of the Trichoderma harzianum wettable powder can obviously improve the germination index, the germination vigor, the embryonic root length and the embryonic bud length of tomato seeds by 100.3 percent, 32 percent, 142.3 percent and 86.8 percent respectively compared with a control.
The Trichoderma harzianum wettable powder 100-10000 times liquid seed soaking treatment can obviously improve the plant growth of the tomato, wherein the maximum growth rates of the plant height, the root length, the stem thickness and the fresh weight of the plant are respectively 8.4-10.7%, 11.0-27%, 8.2-10.6% and 7.5-34.3%, but the difference among treatment groups is small.
The Trichoderma harzianum wettable powder 100-fold liquid 1000-fold is used for root irrigation treatment, the plant growth of the tomato can be obviously improved, and the effect of the 100-fold liquid and the 1000-fold liquid has no obvious difference. Compared with the control, the tomato plant treated by root irrigation has the increases of plant height, root length, stem thickness and fresh weight of the plant of 20.4-34.3%, 4.1-6.2%, 8.3-21.1% and 25.1-81.6% respectively.
2. According to the invention, household garbage, weed fallen leaves or cheap agricultural wastes with low application value are used as raw materials, the fermentation material available for trichoderma harzianum is screened and added into the original formula, so that the fermentation biomass is improved, the use of wheat bran is reduced, the fermentation cost is reduced, the wastes are utilized, and the environment is protected.
3. The powder has the double effects of promoting growth and preventing and treating diseases, has the advantages of high potency, long storage period, convenient application, stable effect and the like, and has the effects of biological fertilizer and biological pesticide.
Drawings
FIG. 1 shows the effect of Trichoderma harzianum wettable powder seed soaking treatment on tomato plant growth vigor. Wherein 1 is the treatment of 100 times of the wettable powder of trichoderma harzianum; 2, processing 1000 times of the wettable powder of trichoderma harzianum by using liquid; 3, processing the Trichoderma harzianum wettable powder by 10000 times of solution; control treatment 4.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the trichoderma harzianum fermentation process of the embodiment is carried out according to the following steps:
firstly, selecting and processing raw materials: selecting domestic garbage or agricultural wastes as raw materials, drying and then crushing for later use by a crusher; the domestic garbage comprises folium sarcandrae, grape leaves and pineapple peels, and the agricultural wastes comprise corncobs and wheat bran;
secondly, preparing a trichoderma harzianum solid state fermentation culture medium: adding the sardine leaves, the grape leaves, the corncobs, the pineapple peels and the wheat bran into a container to obtain a trichoderma harzianum solid state fermentation culture medium; the mass ratio of the Chinese hawthorn leaf to the grape leaf to the corn cob to the pineapple peel to the wheat bran is (3-6): 1-2): 20-41;
and thirdly, fermentation of trichoderma harzianum: and (3) adding the Trichoderma harzianum WY-1 bacterial liquid into the Trichoderma harzianum solid fermentation culture medium prepared in the step two, wherein the culture conditions are as follows: the initial water content is 80-100%, the inoculation amount is 6-10%, and the fermentation of trichoderma harzianum is completed after 10d-12d of culture at 25-28 ℃ to obtain conidium powder; the preparation method of the trichoderma harzianum liquid comprises the following steps: picking and inoculating Trichoderma harzianum WY-1 into a potato glucose culture medium, and carrying out shake flask culture at 25-28 ℃ for 2d-4d, wherein the Trichoderma harzianum WY-1 is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation date of 2016, 4 and 11 days and the preservation number of CGMCC No. 12165.
The second embodiment is as follows: the difference between the first embodiment and the second embodiment is that the mass ratio of the malus baccata leaves, the grape leaves, the corn cobs, the pineapple peels and the wheat bran in the second embodiment is 4:4:4:1: 27. Other steps and parameters are the same as those in the first embodiment.
The third concrete implementation mode: the difference between this embodiment and the first embodiment is that the culture conditions in the third step are: the initial water content is 100%, the inoculation amount is 7%, and the fermentation of the trichoderma harzianum is completed after 10 days of culture at 28 ℃. Other steps and parameters are the same as those in the first embodiment.
The fourth concrete implementation mode: the wettable powder prepared from the conidium powder obtained by the trichoderma harzianum fermentation process comprises 1% -2% of the conidium powder obtained by trichoderma harzianum fermentation, 5% -8% of a wetting agent, 3% -6% of a dispersing agent and the balance of a carrier according to the mass fraction; the wetting agent is one or a composition of more of Sodium Dodecyl Benzene Sulfonate (SDBS), nekal, fatty alcohol-polyoxyethylene ether (JFC) and tween-80; the dispersing agent is one or a composition of more of sodium lignosulphonate (M9), 2-naphthalene sulfonic acid formaldehyde polymer sodium salt (NNO), carboxymethyl cellulose (CMC) and polyethylene glycol 10000(PEG 10000); the carrier is one or a combination of more of kaolin, diatomite, talcum powder, Ca-based bentonite, attapulgite and zeolite powder.
The fifth concrete implementation mode: the difference between the embodiment and the fourth embodiment is that 1-2% of potato juice and 1-2% of glycerin are respectively added as an auxiliary agent during application of the prepared wettable powder. The other steps and parameters are the same as those in the fourth embodiment.
The sixth specific implementation mode: the wettable powder prepared by the fourth specific embodiment has the effect of promoting germination of tomato seeds and has obvious control effects on powdery mildew, gray mold and leaf mold of tomatoes.
The seventh embodiment: the fifth difference from the specific embodiment is that the 100-10000-fold liquid of the Trichoderma harzianum wettable powder has an obvious effect of promoting germination of tomato seeds, and the 1000-10000-fold liquid of the Trichoderma harzianum wettable powder has an obvious effect of preventing and treating powdery mildew, gray mold and leaf mold of tomato. The other steps and parameters are the same as those in the fifth embodiment.
Example 1
The trichoderma harzianum fermentation process of the embodiment is carried out according to the following steps:
the method is characterized in that domestic garbage, agricultural wastes, patrinia scabiosaefolia, wild weeds and the like are used as raw materials, and available fermentation materials are screened. The collected raw materials are placed in an oven to be dried at 60 ℃, and then are crushed by a crusher for standby. Adding 4g of sardine leaves, 4g of grape leaves, 4g of corncobs, 1g of pineapple peels and 27g of wheat bran into a container to obtain a trichoderma harzianum solid state fermentation culture medium; then adding 5ml distilled water, mixing well and autoclaving for 20min, cooling and repeating the sterilization once more. Adding the Trichoderma harzianum WY-1 bacterial liquid into a Trichoderma harzianum solid state fermentation culture medium, wherein the culture conditions are as follows: the initial water content is 100%, the inoculation amount is 7%, and the fermentation of trichoderma harzianum is completed after 10 days of culture at 28 ℃ to obtain conidium powder; the preparation method of the trichoderma harzianum liquid comprises the following steps: trichoderma harzianum WY-1 was picked and inoculated into potato dextrose medium, and shake-flask culture was carried out at 28 ℃ for 3 d. And 10d, taking out the fermentation product, drying the fermentation product in an oven at 40 ℃ for 12h, crushing the fermentation product by using a crusher, sieving the fermentation product by using a 200-mesh sieve, and measuring the concentration of the live spores of the fermentation product by using a gradient dilution method. The spore yield of the embodiment can reach 2.79 multiplied by 1011CFU/g。
Example 2
Candidate carriers are kaolin, diatomite, talcum powder, bentonite, attapulgite and zeolite powder, candidate wetting agents are SDBS, nekal, JFC and tween-80, candidate dispersing agents are M9, NNO, CMC and PEG10000, the candidate dispersing agents are all crushed and sieved by a 200-mesh sieve after being sterilized and dried, the crushed candidate dispersing agents are respectively added into a culture medium, and the influence of the carriers on the growth speed of trichoderma harzianum and the germination rate of conidia is measured. After primary screening, the carrier was mixed with 10% of fine powder of spores (200 mesh) screened, and the wetting time and suspension rate were measured. The wettability and the suspensibility refer to a method for measuring the wettability and the suspensibility of GB/T14825-93 pesticide wettable powder and are slightly modified.
1) Preparing standard hard water: 0.304g of anhydrous calcium chloride and 0.139g of magnesium chloride with crystal water are weighed, dissolved in 600mL of water in a 1000mL beaker, and poured into a 100mL volumetric flask. The beaker was rinsed with 300mL portions of water, the rinse solution was poured into the flask together, and the flask was made to hold a constant volume with water and shaken well (see GB/T5451-2001).
2) And (3) wettability determination: 100ml of 342mg/L standard hard water is taken and poured into a 250ml beaker, and the beaker is placed in a thermostatic water bath at 25 ℃ so that the liquid level of the beaker is flush with the liquid level of the water bath. Weighing 5g of sample, uniformly pouring the sample on the liquid surface of the beaker from a position which is level with the opening of the beaker, immediately timing by using a stopwatch during sample adding until the sample is completely wetted, recording the wetting time, and repeatedly measuring for 5 times to obtain an average value.
3) Suspension property determination: the method for determining the suspension percentage of the pesticide wettable powder is slightly changed with reference to GB/T14825-93: weighing 0.1g of sample, placing the sample in a centrifuge tube (50ml with a cover) filled with 10ml of standard hard water, shaking uniformly, diluting the sample to 25ml with water, covering the centrifuge tube, taking the bottom of the centrifuge tube as an axis, turning the centrifuge tube upside down for 30 times within 1min, opening the cover, standing the centrifuge tube in a water bath at 30 ℃ for 30min, slightly sucking 1/10 liquid by a suction tube, and measuring the content of active ingredients in the liquid.
The carrier and the surfactant are properly screened out through a biocompatibility experiment, the addition amount of the carrier and the surfactant is determined by comparing wettability and suspension property and comprehensively considering the performance, source, price and other factors of the surfactant, and the formula of the Trichoderma harzianum wettable powder is 1% of spore powder, 6% of Tween-80, 4% of NNO and talcum powder (filling). The inventive synergist is matched with the preparation for use, the synergist X has a synergistic ratio of 69.23 percent and an obvious effect (figure 1) by taking powdery mildew as an object.
The preparation of the wettable powder of the embodiment is carried out according to the following steps:
inoculating Trichoderma harzianum pure bacteria into PDA culture medium, culturing at 28 deg.C for 10 days to obtain spore with concentration of 108Inoculating a CFU/ml spore suspension into a culture bottle according to the inoculation amount of 4%, performing shake culture at 180rpm for 3d to serve as seed liquid, inoculating into a solid fermentation substrate, performing culture at 28 ℃ for 10d, drying at 40 ℃, crushing by using a universal crusher, sieving by using a 200-mesh sieve, and collecting trichoderma harzianum conidium powder. The amount of viable spores of the conidium powder was measured and left at 4 ℃ for further use. And adding 1g of the obtained conidium powder into a carrier, 5g of a wetting agent and 5g of a dispersing agent to prepare the trichoderma harzianum conidium powder. The concentration of the conidium powder of trichoderma harzianum is 1000 times of the solution, and 1% of potato juice and 1% of glycerol are respectively added as an auxiliary agent during application.
Growth-promoting effect of Trichoderma harzianum wettable powder
Diluting trichoderma harzianum powder at different concentrations, performing seed soaking treatment on tomato seeds, performing root irrigation treatment on tomato seedlings, exploring the growth promoting effect of microbial inoculum seed soaking and root irrigation on the tomato seeds and the tomato seedlings, and determining the appropriate seed soaking and root irrigation concentrations. The result shows that the seed soaking of the trichoderma harzianum spore liquid has the effect of promoting the germination of tomato seeds, and the spore concentration is 1/2 multiplied by 107CFU/ml, the best effect. The tomato plant growth promoting effect is achieved by the bacterial agent seed soaking and root irrigation treatment, and the optimal concentration is 1000 times of the bacterial agent diluent (table 1).
TABLE 1 Effect of different microbial inoculum concentrations on growth of tomato plants
Control effect of trichoderma harzianum (II) on common tomato diseases
The trichoderma powder is diluted in different concentrations and used for preventing and treating common tomato diseases (gray mold, leaf mold and powdery mildew), and the prepared microbial inoculum disease prevention effect is researched by taking a chemical agent (80% carbendazim wettable powder) and commercially available trichoderma (3 hundred million/g trichoderma harzianum wettable powder in Baowo USA) as a reference, and the appropriate use concentration is obtained. The Trichoderma harzianum wettable powder has obvious control effects on powdery mildew of tomato cultivated in greenhouse, gray mold and leaf mold of indoor potted tomato, and the 1000-time liquid effect of the microbial inoculum is the most obvious (tables 2 and 3).
TABLE 2 prevention and control effect of Trichoderma harzianum wettable powder on gray mold of indoor potted tomato
TABLE 3 control effect of Trichoderma harzianum wettable powder on indoor potted tomato leaf mold
Claims (4)
1. A fermentation process of trichoderma harzianum is characterized in that: the process comprises the following steps:
firstly, selecting and processing raw materials: selecting domestic garbage or agricultural wastes as raw materials, drying and then crushing for later use by a crusher; the domestic garbage comprises folium sarcandrae, grape leaves and pineapple peels, and the agricultural wastes comprise corncobs and wheat bran;
secondly, preparing a trichoderma harzianum solid state fermentation culture medium: adding the sardine leaves, the grape leaves, the corncobs, the pineapple peels and the wheat bran into a container to obtain a trichoderma harzianum solid state fermentation culture medium; the mass ratio of the Chinese wild rye leaves to the grape leaves to the corncobs to the pineapple peels to the wheat bran is 4:4:4:1: 27;
and thirdly, fermentation of trichoderma harzianum: and (3) adding the Trichoderma harzianum WY-1 bacterial liquid into the Trichoderma harzianum solid fermentation culture medium prepared in the step two, wherein the culture conditions are as follows: the initial water content is 100%, the inoculation amount is 7%, and the fermentation of trichoderma harzianum is completed after 10 days of culture at 28 ℃ to obtain conidium powder; the preparation method of the trichoderma harzianum liquid comprises the following steps: picking and inoculating Trichoderma harzianum WY-1 into a potato glucose culture medium, and carrying out shake flask culture at 25-28 ℃ for 2d-4d, wherein the Trichoderma harzianum WY-1 is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation date of 2016, 4 and 11 days and the preservation number of CGMCC No. 12165.
2. The application of the wettable powder prepared from the conidium powder obtained by the trichoderma harzianum fermentation process in claim 1 is characterized in that: the wettable powder has the function of promoting germination of tomato seeds and has the function of preventing and treating powdery mildew, gray mold and leaf mold of tomatoes; the preparation method of the wettable powder comprises the following steps: the wettable powder comprises 1 to 2 mass percent of conidium powder obtained by fermenting trichoderma harzianum, 5 to 8 mass percent of wetting agent, 3 to 6 mass percent of dispersing agent and the balance of carrier; the wetting agent is one or a composition of more of sodium dodecyl benzene sulfonate, nekal, fatty alcohol-polyoxyethylene ether and tween-80; the dispersing agent is one or a composition of more of sodium lignosulphonate, 2-naphthalene sulfonic acid formaldehyde polymer sodium salt, carboxymethyl cellulose and polyethylene glycol 10000; the carrier is one or a combination of more of kaolin, diatomite, talcum powder, Ca-based bentonite, attapulgite and zeolite powder.
3. The application of the wettable powder prepared from the conidiospore powder obtained by the trichoderma harzianum fermentation process according to claim 2 is characterized in that: 1-2% of potato juice and 1-2% of glycerin are respectively added as an auxiliary agent during application of the wettable powder.
4. The application of the wettable powder prepared from the conidiospore powder obtained by the trichoderma harzianum fermentation process according to claim 2 is characterized in that: the 100-10000-fold liquid of the Trichoderma harzianum wettable powder has the effect of promoting germination of tomato seeds, and the 1000-10000-fold liquid of the Trichoderma harzianum wettable powder has the effect of preventing and treating powdery mildew, gray mold and leaf mold of tomatoes.
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