Tomato seedling microbial fertilizer for preventing and treating pathogenic fungi as well as preparation and application of tomato seedling microbial fertilizer
By preparing tomato seedling microbial fertilizer, using the combination of compost and Bacillus vegetarian solution, environmental pollution and food safety issues of traditional chemical control methods are solved, and effective antagonism and resource utilization of various pathogenic fungi are achieved.
Patent Information
- Application Number
- CN202411511506.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2025-02-07
AI Technical Summary
Traditional methods of preventing and controlling soil-borne diseases mainly rely on chemical methods, which lead to environmental pollution and human health hazards, and it is difficult to effectively prevent and control a variety of pathogenic fungi.
By preparing a microbial fertilizer for tomato seedlings to prevent and treat pathogenic fungi, compost with tomato seedlings and corn stalks, and inoculated with antibiotic-labeled Bacillus Bacillus Bacillus bacteria solution, secondary fermentation was performed to prepare microbial fertilizer with antagonistic effect.
This method not only has a good antagonistic effect on common pathogenic fungi in the soil, but also effectively solves the resource utilization problem of tomato seedlings, which is of great significance to people's food safety.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of organic fertilizer preparation, and in particular to a tomato seedling microbial fertilizer for preventing and controlling pathogenic fungi, and a preparation method and application thereof. Background Art
[0002] About 20% of the world's crop yields are lost due to plant diseases, of which about 70% are caused by soil-borne fungal infections. Soil-borne fungal diseases are highly concealed, contagious, explosive and destructive, and can also be spread through air, rain, irrigation, agricultural operations, etc. Under suitable temperature and humidity conditions, the disease will cause epidemics and outbreaks in a short period of time. The main pathogen of cucumber wilt is Fusarium oxysporum cucumber-specific type, which can occur at all stages of cucumber growth. The pathogen can be spread through the soil and can also directly attack other structures of the plant, such as stems and leaves. Watermelon wilt is one of the most serious watermelon diseases. It can occur throughout the entire growing period of watermelon, causing a decrease in watermelon yield, or even a total loss of yield in severe cases, resulting in huge economic losses and waste of resources in watermelon production. Soil-borne fungi can survive in harsh environments such as soil or crop residues in the form of resistant reproductive bodies such as sclerotia and conidia. They have a wide host range and kill host tissues by secreting degradative enzymes or toxins, and produce mycelium to absorb nutrients from the host, causing plants to wilt, become ill or necrotize.
[0003] Traditional methods of preventing and controlling soil-borne diseases mostly use chemical methods, but since chemical control causes serious environmental pollution and harms human health, advocating biological control is conducive to sustainable economic development. Microbial fertilizers can not only inhibit the growth and reproduction of pathogens, but also provide sufficient nutrients for plant growth.
[0004] Therefore, the present invention provides a tomato seedling microbial fertilizer for preventing and controlling pathogenic fungi and its preparation and application, which better combines biocontrol bacteria and waste, provides a theoretical basis and technical support for the research and development of microbial fertilizers for preventing and controlling various pathogenic fungi, and is of great significance to people's food safety. Summary of the invention
[0005] The purpose of the invention is to provide a tomato seedling microbial fertilizer for preventing and controlling pathogenic fungi and its preparation and application, so as to fill the gap in the current technology.
[0006] The purpose of the present invention is achieved through the following technical solutions:
[0007] A method for preparing a tomato seedling microbial fertilizer for preventing and controlling pathogenic fungi comprises the following steps:
[0008] (1) Preparation of tomato vine compost:
[0009] Harvesting tomato vines and corn stalks, crushing the tomato vines and corn stalks into small segments of 10 mm, mixing the small segments of tomato vines and corn stalks to adjust the C / N and moisture content, placing them in an automatic constant temperature box, inoculating them with a decomposition-promoting bacterial agent and cellulose hydrolyzing enzyme, ventilating them for composting, and using an automatic temperature detector to detect the temperature change in real time, turning the compost every 2 to 4 days, and ending the composting process after 15 days for use;
[0010] (2) Preparation of antibiotic-labeled Bacillus Velez bacteria solution:
[0011] A single colony of Escherichia coli with a chloramphenicol resistance gene is picked, streaked on a resistance plate with a chloramphenicol concentration of 5 mg / ml, and placed in a 37°C incubator for 16 to 24 hours, a single colony is picked from the plate and placed in an LB liquid culture medium with a concentration of 5 mg / ml, the test tube is placed in a shaker for culturing, and the resistance gene is extracted using a plasmid mini-extraction kit; competent cells are prepared from wild Bacillus Velez (BER1), the chloramphenicol resistance gene is introduced into the competent cells of Bacillus Velez, and then the plates are coated, and the plates are placed in a 37°C incubator for culturing for 16 to 24 hours, a single colony is picked from the plate, and a Bacillus Velez bacterial liquid is prepared until the logarithmic growth phase, and the labeled Bacillus Velez bacterial liquid is stored in a -80°C glycerol tube;
[0012] (3) Preparation of tomato seedling microbial fertilizer:
[0013] The Bacillus Velez subtilis liquid prepared in step (2) is inoculated into the tomato vine compost, placed in a 35° C. incubator for secondary fermentation for 30 days, and the dynamic changes of the number of live bacteria and the number of spores are monitored to obtain a tomato vine microbial fertilizer.
[0014] Preferably, in step (1), the tomato vine segments and corn stalk segments are mixed to adjust the C / N ratio to 20 to 40:1 and the moisture content to 55% to 65%.
[0015] Preferably, in step (1), the temperature of the automatic thermostat is 50-60°C, and the ventilation is 200-220m 3 / h for composting.
[0016] Preferably, in step (2), the LB liquid culture medium comprises yeast extract, tryptone, NaCl, and distilled water, and the pH is adjusted to 7.0.
[0017] Preferably, in step (2), the shaking incubation conditions are 200-210 rpm and 37° C. for 24-28 hours.
[0018] Preferably, in step (2), the viable bacterial count of the prepared Bacillus velezinsis liquid is 1.03×10 9 CFU / mL.
[0019] Preferably, in the step (3), the prepared Bacillus Velez subtilis liquid is inoculated into the tomato seedling compost at a ratio of 10%.
[0020] The present application also claims protection for a tomato vine microbial fertilizer for preventing and controlling pathogenic fungi prepared by the above-mentioned method for preparing the tomato vine microbial fertilizer for preventing and controlling pathogenic fungi.
[0021] The present application also claims to protect the use of the above-mentioned tomato vine microbial fertilizer for controlling pathogenic fungi in plant disease prevention.
[0022] Due to the application of the above technical solution, the present invention has the following beneficial effects compared with the prior art:
[0023] 1. The present invention provides a method for preparing a microbial fertilizer for preventing and controlling a variety of pathogenic fungi, which not only has a good antagonistic effect on common pathogenic fungi in the soil but also effectively solves the problem of resource utilization of tomato seedlings;
[0024] 2. The present invention better combines biocontrol bacteria with waste, provides a theoretical basis and technical support for the research and development of microbial fertilizers for preventing and controlling a variety of pathogenic fungi, and is of great significance to people's food safety. DETAILED DESCRIPTION
[0025] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, a specific implementation scheme is now described in detail.
[0026] The present invention is further described below in conjunction with the examples, but the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not indicated are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.
[0027] Example 1
[0028] This embodiment provides a method for preparing a tomato seedling microbial fertilizer for preventing and controlling pathogenic fungi, comprising the following steps:
[0029] (1) Preparation of tomato vine compost:
[0030] Harvest tomato vines and corn stalks, crush them into 10 mm small segments, mix the small segments of tomato vines and corn stalks to adjust the C / N ratio to 25 and the moisture content to 60%, put them into a 50℃ automatic constant temperature box, inoculate them with a decomposition-promoting bacterial agent and cellulose hydrolyzing enzyme, and ventilate for 200 m 3 / h to compost, and use an automatic temperature detector to detect temperature changes in real time, turn the compost every 3 days, and stop after 15 days for standby use;
[0031] (2) Preparation of antibiotic-labeled Bacillus Velez bacteria solution:
[0032] A single colony of Escherichia coli with a chloramphenicol resistance gene was picked, streaked on a resistance plate with a chloramphenicol concentration of 5 mg / ml and placed in a 37°C incubator for 24 hours, a single colony was picked from the plate and placed in 5 ml of LB liquid culture medium with a concentration of 5 mg / ml (the liquid culture medium includes 5 g of yeast extract, 10 g of tryptone, 10 g of NaCl, 1000 mL of distilled water, and the pH is adjusted to 7.0), the test tube was placed in a 37°C, 200 rpm shaker for 24 hours, and the resistance gene was extracted using a plasmid mini-extraction kit; competent cells were prepared from wild Bacillus Velez (BER1), the chloramphenicol resistance gene was introduced into the competent cells of Bacillus Velez, and the plates were coated and placed in a 37°C incubator for 24 hours, a single colony was picked from the plate, and the culture was continued until the logarithmic growth phase, a Bacillus Velez bacterial solution was prepared, and the labeled Bacillus Velez was stored in a -80°C glycerol tube, and the number of viable cells was 1.03×10 9 CFU / mL;
[0033] (3) Preparation of tomato seedling microbial fertilizer:
[0034] The Bacillus Velez subtilis liquid prepared in step (2) was inoculated into the tomato seedling compost at an inoculation ratio of 10%, and placed in a 35°C incubator for secondary fermentation for 30 days. The dynamic changes of the number of live bacteria and the number of spores were monitored to obtain a tomato seedling microbial fertilizer. The effective number of live bacteria was determined by the plate count method to be 4.86×10 7 CFU / mL.
[0035] The tomato vine microbial fertilizer obtained by the preparation method of the tomato vine microbial fertilizer for preventing and controlling pathogenic fungi is used to prevent and control cucumber wilt, watermelon wilt and tomato bacterial wilt, as follows:
[0036] ① Potted experiment on the prevention and treatment of cucumber wilt by tomato vine microbial fertilizer:
[0037] Mix the tomato poplar microbial fertilizer and chemical fertilizer with the matrix soil at a ratio of 2%, take the cucumber seedlings with 2 leaves and 1 heart and transplant them, insert a sterile blade into the soil 1-2 cm away from the base of the cucumber stem and 2-3 cm to injure the root, then add 1×10 6 cfu / mL was injected into the cucumber roots with a sterile syringe, 10mL per seedling, and 10mL of fermented bacterial solution was used to irrigate the roots 3 days later; the substrate soil without fertilizer was used as a blank control; each treatment was repeated 3 times, with 8 seedlings per repeat, and the disease was investigated after 14 days, and the disease index and control effect were calculated. The experiment was repeated twice;
[0038] Cucumber wilt grading standard: Grade 0, no symptoms; Grade 1, yellowing or wilting area of true leaves and cotyledons does not exceed 50% of the total area; Grade 2: yellowing or wilting area of true leaves and cotyledons exceeds 50% of the total area; Grade 3: leaves wilt or die, and only the growing points survive; Grade 4: the whole plant severely wilts and even dies. Jiang Dongyang et al.: Identification of antagonistic actinomycetes 2-1-2F and its efficacy in preventing cucumber wilt 415
[0039] ② Potted experiment on the prevention and treatment of watermelon wilt by tomato seedling microbial fertilizer:
[0040] The specification of the cultivation pot was 385mm×285mm, and each pot was filled with 9.0kg of soil. Microbial fertilizer for tomato seedlings and chemical fertilizer were mixed with the matrix soil at a ratio of 2% respectively; the matrix soil without fertilizer was used as a blank control; each treatment was repeated 3 times, and healthy watermelon seedlings with good growth conditions and uniform size were selected. After the seedlings grew 3 to 4 true leaves, they were transplanted into pots. After 10 days of transplanting, the bacterial suspension of watermelon wilt pathogen was inoculated into each pot to reach 10 6 CFU / mL.
[0041] ③ Potted experiment on the prevention and treatment of tomato bacterial wilt by using tomato seedling microbial fertilizer:
[0042] Seedling cultivation: Take tomato seeds, soak them in sterile distilled water for 24 hours, and then soak them in warm water at 55°C for 5 minutes, so that the seeds can absorb sufficient water and be more conducive to germination; then spread two layers of gauze on a clean tray, add sterile water to the tray, use tweezers to put the tomato seeds into the tray one by one, and then place the tray in a constant temperature incubator at 28°C and dark conditions to germinate for 1-2 days; transplant the tomato seeds that have sprouted into a seedling tray containing substrate soil, and place it in a constant temperature light incubator at 28°C and 16h light for about 1-2 days for use;
[0043] Potted plants: Mix the microbial fertilizer and chemical fertilizer with the matrix soil at a ratio of 2%, and use the matrix soil without fertilizer as a blank control; transfer the seedlings of similar growth size in the seedling tray to the pots filled with matrix soil, and add 10mL (10 8 CFU / mL) were poured into the flower pots. The pot experiment was carried out in a greenhouse at a temperature of 24-28°C, a RH of 75-90%, and a photoperiod of 12h. Each treatment consisted of 5 plants, and the experiment was repeated 3 times. The severity of the disease was assessed 28 days after inoculation.
[0044] The disease index is divided into 0-4: 0, no wilting; 1, 1-25% of the leaves are wilted; 2, 25-50% of the leaves are wilted; 3, 51-75% of the leaves are wilted; 4, 76-100% of the leaves are wilted.
[0045] The statistical methods of disease index and control effect are as follows:
[0046] Disease index = Σ(number of diseased plants × disease level) / (total number of plants × highest level) × 100%
[0047] The control effect = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100%.
[0048] The disease prevention effect of the above-mentioned tomato vine microbial fertilizer for preventing and controlling cucumber wilt, watermelon wilt and tomato bacterial wilt is shown in Table 1.
[0049] Table 1
[0050]
[0051] It can be seen from Table 1 that, compared with the blank control treatment and the chemical fertilizer treatment, the tomato vine microbial fertilizer has a good inhibitory effect on cucumber wilt, watermelon wilt and tomato bacterial wilt, and the inhibitory effect on the three pathogens can reach more than 50%.
[0052] In summary, the present invention provides a method for preparing a microbial fertilizer for preventing and controlling a variety of pathogenic fungi, which not only has a good antagonistic effect on common pathogenic fungi in the soil but also effectively solves the problem of resource utilization of tomato seedlings; the present invention better combines biocontrol bacteria and waste, provides a theoretical basis and technical support for the research and development of microbial fertilizers for preventing and controlling a variety of pathogenic fungi, and is of great significance to people's food safety.
[0053] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.
Claims
1. A method for preparing a tomato seedling microbial fertilizer for preventing and controlling pathogenic fungi, characterized in that: The steps include: (1) Preparation of tomato vine compost: Harvesting tomato vines and corn stalks, crushing the tomato vines and corn stalks into small segments of 10 mm, mixing the small segments of tomato vines and corn stalks to adjust the C / N and moisture content, placing them in an automatic constant temperature box, inoculating them with a decomposition-promoting bacterial agent and cellulose hydrolyzing enzyme, ventilating them for composting, and using an automatic temperature detector to detect the temperature change in real time, turning the compost every 2 to 4 days, and ending the composting process after 15 days for use; (2) Preparation of antibiotic-labeled Bacillus Velez bacteria solution: A single colony of Escherichia coli with a chloramphenicol resistance gene is picked, streaked on a resistance plate with a chloramphenicol concentration of 5 mg / ml, and placed in a 37°C incubator for 16 to 24 hours. A single colony is picked from the plate and placed in an LB liquid culture medium with a concentration of 5 mg / ml. The test tube is placed in a shaker for culturing, and then the resistance gene is extracted using a plasmid extraction kit. Competent cells are prepared from wild Bacillus Velezii bacteria, the chloramphenicol resistance gene is introduced into the competent cells of Bacillus Velezii, and then the plates are coated and placed in a 37°C incubator for culturing 16 to 24 hours. A single colony is picked from the plate until the logarithmic growth phase, and a Bacillus Velezii bacterial liquid is prepared, and the labeled Bacillus Velezii is stored in a -80°C glycerol tube; (3) Preparation of tomato seedling microbial fertilizer: The Bacillus Velez subtilis liquid prepared in step (2) is inoculated into the tomato vine compost, placed in a 35° C. incubator for secondary fermentation for 30 days, and the dynamic changes of the number of live bacteria and the number of spores are monitored to obtain a tomato vine microbial fertilizer.
2. The method for preparing a microbial fertilizer for tomato seedlings for controlling pathogenic fungi according to claim 1, characterized in that: In the step (1), the tomato vine segments and corn stalk segments are mixed to adjust the C / N ratio to 20-40:1 and the moisture content to 55%-65%.
3. The method for preparing a microbial fertilizer for tomato seedlings for controlling pathogenic fungi according to claim 1, characterized in that: In the step (1), the temperature of the automatic thermostat is 50-60°C, and the ventilation is 200-220m 3 / h for composting.
4. The method for preparing a microbial fertilizer for tomato seedlings for controlling pathogenic fungi according to claim 1, characterized in that: In the step (2), the LB liquid culture medium includes yeast extract, tryptone, NaCl, and distilled water, and the pH is adjusted to 7.
0.
5. The method for preparing a microbial fertilizer for tomato seedlings for controlling pathogenic fungi according to claim 1, characterized in that: In the step (2), the shaking incubation condition is 200-210 rpm and 37° C. for 24-28 hours.
6. The method for preparing a microbial fertilizer for tomato seedlings for controlling pathogenic fungi according to claim 1, characterized in that: In the step (2), the viable bacterial count of the prepared Bacillus Velezii liquid was 1.03×10 9 CFU / mL.
7. The method for preparing a microbial fertilizer for tomato seedlings for controlling pathogenic fungi according to claim 1, characterized in that: In the step (3), the prepared Bacillus Velez subtilis liquid is inoculated into the tomato seedling compost at a ratio of 10%.
8. A tomato seedling microbial fertilizer for controlling pathogenic fungi prepared by the method for preparing the tomato seedling microbial fertilizer for controlling pathogenic fungi as claimed in any one of claims 1 to 7.
9. Use of the tomato seedling microbial fertilizer for controlling pathogenic fungi prepared by the preparation method of the tomato seedling microbial fertilizer for controlling pathogenic fungi as claimed in any one of claims 1 to 7 or the tomato seedling microbial fertilizer for controlling pathogenic fungi as claimed in claim 8 in plant disease prevention.