A production scheme of a microbial fertilizer of bacillus thuringiensis for tea garden

Abstract

The application provides a production scheme of a tea garden self-nitrogen-fixing bacillus thuringiensis microbial fertilizer, and specifically discloses a method for producing a tea garden microbial fertilizer by using self-nitrogen-fixing bacillus thuringiensis.The fermentation liquor of the self-nitrogen-fixing bacillus thuringiensis has a promoting effect on the growth of the root, stem, fresh weight and chlorophyll content of tea seedlings, so that a non-antagonistic strain is selected to produce a microbial fertilizer, and the microbial fertilizer is tested according to national standards.The application provides a theoretical basis for diversified application of bacillus thuringiensis, and the concept of shared use of the microbial fertilizer and pesticides has a remarkable economic effect.

Description

[0001] Production plan Technical Field

[0002] This invention belongs to the field of pesticide technology, specifically relating to a method for producing a self-generating nitrogen-fixing Bacillus thuringiensis microbial fertilizer for tea gardens. Background Technology

[0003] Nitrogen is an essential element for plant growth. Applying nitrogen fertilizer can effectively increase tea yield and the content of free amino acids, tea polyphenols, and caffeine in tea leaves. Since 2010, high-yield tea gardens have applied an annual nitrogen application rate of 450 kg / ha. -2 The primary fertilizer used is chemical fertilizer, while organic fertilizer accounts for less than 30%. The excessive use of chemical nitrogen fertilizer accelerates soil acidification and the emission of nitrogen oxides (N2O and NO). Nitrous oxide (N2O) is a greenhouse gas, and its ozone-depleting effect is 265 times that of carbon dioxide (CO2). The optimal soil pH for tea tree growth is approximately 4.5-5.5; soil samples from 46% of tea-producing areas in China have a pH below 4.5, low nitrogen content, and low organic matter content. In 2017, nitrogen loss from agriculture reached 719,500 tons, accounting for 23.7% of total nitrogen pollution emissions. Reducing chemical nitrogen fertilizer use and adjusting fertilizer types are crucial to reducing nitrogen emissions and pollution. Compared to chemical fertilizers, the nitrogen-fixing microorganisms in microbial nitrogen fertilizers fix active nitrogen in the soil, effectively improving soil structure, enhancing soil fertility, promoting plant growth and development, improving crop quality, and strengthening crop resistance to pests and diseases. This mitigates the harm of excessive chemical fertilizer use and ensures green and sustainable agricultural development. Summary of the Invention

[0004] To address existing problems, this invention relates to a method for producing a self-generating nitrogen-fixing Bacillus thuringiensis microbial fertilizer for tea gardens. The raw materials used in this invention are inexpensive, the production method is simple, and the microorganisms used not only promote the growth of tea seedlings but also meet national standards.

[0005] The technical solution adopted in this invention is as follows: A method for producing a self-generating nitrogen-fixing Bacillus thuringiensis microbial fertilizer for tea gardens, the raw materials used include: potassium chloride, potassium dihydrogen phosphate, calcium ammonium nitrate, zinc sulfate heptahydrate, diammonium phosphate and humic acid (HA), the added materials include one or more of the following: carbon and nitrogen source mixture, trace elements, corn steep liquor, defoamer, wood ash and nutrient solution.

[0006] The effect of fermentation broth of Bacillus thuringiensis, a nitrogen-fixing bacterium, on the growth of tea seedlings; Antagonism test of autotrophic nitrogen-fixing Bacillus thuringiensis; Production of self-generating nitrogen-fixing Bacillus thuringiensis microbial fertilizer.

[0007] Methods for testing the effect of Bacillus thuringiensis fermentation broth on tea seedling growth: Purchase tea seedlings (Fuding Dahao). Select three seedlings with minimal growth error as a group. Measure the seedling height, root length, fresh weight, and chlorophyll content of each seedling, record the data, and then plant them. Allow them to grow for one week to ensure survival. Activate the test strain overnight for 12 hours. Transfer 100 μL of the bacterial solution to a shake flask and culture for 36 hours. Dilute 10 mL of the bacterial solution 100 times and use it to irrigate the tea seedlings. Repeat this process every 15 days. After three months, measure the seedling height, root length, fresh weight, and chlorophyll content again and calculate the average net growth value.

[0008] Method for testing the antagonistic effect of free-living nitrogen-fixing Bacillus thuringiensis: Three free-living nitrogen-fixing Bt strains were selected and grouped into four groups (two-by-two and three-by-three). The strains were activated for 12 h and the bacterial solution was diluted to 10⁻⁶. -5 Take 100 μL of bacterial suspension and spread it evenly on LB solid medium. Incubate at 30℃ for 12 h. Select single colonies and streak them crosswise according to the combination method. Incubate at 30℃ for 12 h and observe whether the strains have antagonistic effects.

[0009] Method for preparing Bacillus thuringiensis microbial fertilizer: Activate the strain for 12 h, streak on LB agar plates, pick out single colonies and transfer them to 150 mL of fermentation medium. Incubate at 30 ℃ for 18 h in a constant temperature shaker to obtain seed liquid. Transfer 1% of the seed liquid to 150 mL of fermentation medium and incubate at 30 ℃ for 36 h in a constant temperature shaker to obtain fermentation broth. Mix the fermentation broth of the strains according to 7 different combinations (single, pairwise, and triple combinations) in equal volumes and dispense into 150 mL containers to obtain liquid microbial fertilizer. Take 300 mL of each of the 7 liquid microbial fertilizers and mix them with 1600 g of wood ash and 100 mL of nutrient solution respectively. Pack the mixture into fermentation bags, seal, and ferment for 14 days to obtain solid microbial fertilizer.

[0010] The significant advantages of this invention are: Although Bacillus thuringiensis is widely used in the field of biocontrol, it still has some shortcomings, such as the low retention rate of the strain on the leaves, which leads to the loss of a large amount of effective ingredients in the soil. This solution uses the concept of combining medicine and fertilizer to produce microbial fertilizer, which can not only promote the growth of tea seedlings, but also meet the requirements of national standards. Attached Figure Description

[0011] Figure 1 Effects of fermentation broth of Bacillus thuringiensis, a nitrogen-fixing bacterium, on the fresh weight of tea seedling roots and stems and chlorophyll content.

[0012] Figure 2 Antagonism test of Bacillus thuringiensis, a nitrogen-fixing bacterium.

[0013] Figure 3Production of self-generating nitrogen-fixing Bacillus thuringiensis microbial fertilizer. Detailed Implementation

[0014] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0015] The materials involved in this invention were provided by the Key Laboratory of Biological Pesticides and Chemical Biology of the Ministry of Education of Fujian Agriculture and Forestry University and Fujian Lv'an Biological Pesticide Co., Ltd.

[0016] Example 1: Effects of fermentation broth of Bacillus thuringiensis, a nitrogen-fixing bacterium, on tea seedling growth Three tea seedlings with minimal growth error were selected as a group. Seedling height, root length, fresh weight, and chlorophyll content were measured and recorded. The seedlings were then planted and allowed to grow for one week to ensure survival. The test strain was activated overnight for 12 hours, and 100 μL of the bacterial solution was transferred to a shake flask for 36 hours of culture. 10 mL of the bacterial solution was diluted 100 times and used to irrigate the tea seedlings every 15 days. After three months, seedling height, root length, fresh weight, and chlorophyll content were measured again, and the average net growth value was calculated to observe the effect of the bacterial strain on tea seedling growth.

[0017] The results are as follows Figure 1 As shown: strain BRC-CY1 significantly promoted the growth of root length, stem length, fresh weight and chlorophyll content in tea seedlings; strain HD1 significantly promoted the growth of root length, fresh weight and chlorophyll content in tea seedlings; strain 8010 significantly promoted the growth of fresh weight in tea seedlings.

[0018] Example 2: Antagonistic test of autotrophic nitrogen-fixing Bacillus thuringiensis Three free-living nitrogen-fixing Bt strains were selected and grouped into four groups (two-by-two and three-by-three). The strains were activated for 12 h, and the bacterial solution was diluted 10-5 times. 100 μL of the bacterial solution was taken and evenly spread on LB solid medium and incubated at 30 ℃ for 12 h. Single colonies were selected and streaked crosswise according to the combination method and incubated at 30 ℃ for 12 h to observe whether the strains had antagonistic effects.

[0019] The results are as follows Figure 2 As shown: Different strains were streaked across LB solid medium using different combination methods. There were four combinations of the three strains (HD1, BRC-LJ03 and BRC-CY1) (HD1+BRC-LJ03, HD1+BRC-CY1, BRC-LJ03+BRC-CY1 and HD1+BRC-LJ03+BRC-CY1). All of them could grow crosswise in the center of the LB solid medium without any antagonistic effect between them.

[0020] Example 3: Production of self-growth nitrogen-fixing Bacillus thuringiensis microbial fertilizer The strain was activated for 12 h, streaked on LB agar plates, and single colonies were picked and transferred to 150 mL of fermentation medium. The culture was carried out at 30 °C for 18 h in a constant temperature shaker to obtain seed liquid. 1% of the seed liquid was transferred to 150 mL of fermentation medium prepared with carbon and nitrogen source mixture, trace elements, corn steep liquor, and defoamer. The culture was carried out at 30 °C for 36 h in a constant temperature shaker to obtain fermentation broth. The fermentation broth of the strain was mixed in equal volumes according to 7 schemes, including single combination, pairwise combination, and three-combination, and dispensed into 150 mL containers to obtain liquid microbial fertilizer. 300 mL of each of the 7 liquid microbial fertilizers was taken and mixed evenly with 1600 g of wood ash and 100 mL of nutrient solution, respectively. The mixture was then placed in fermentation bags, sealed, and fermented for 14 days to obtain solid microbial fertilizer.

[0021] The results are as follows Figure 3 As shown: Seven types of liquid and solid microbial fertilizers were prepared by combining three strains (HD1, BRC-LJ03, and BRC-CY1) with no antagonistic effect in different ways. Example 4: National Standard Test of Self-Growing Nitrogen-Fixing Bacillus thuringiensis Microbial Fertilizer Tested in accordance with national standards (GB 20287-2006, GB18877-2002 and GB / T19524.1-2004), including indicators such as viable bacteria count, mold count, miscellaneous bacteria rate, pH, moisture, fineness, fecal coliforms, and shelf life.

[0022] The results are shown in Table 1: The viable count, mold and other microbial count, miscellaneous bacteria rate, pH, moisture content, fineness, fecal coliform count, and shelf life of the seven liquid or solid microbial fertilizers, namely HD1, BRC-LJ03, BRC-CY1, HD1+BRC-LJ03, HD1+BRC-CY1, BRC-LJ03+BRC-CY1, and HD1+BRC-LJ03+BRC-CY1, all met the national standards.

[0023] Table 1: Analysis of National Standard Tests

Claims

1. A method for producing a self-growth nitrogen-fixing Bacillus thuringiensis (Bt) microbial fertilizer for tea gardens, characterized in that: Microbial fertilizers come in both liquid and solid forms; the microorganisms used are Bacillus thuringiensis, a nitrogen-fixing organism.

2. The method according to claim 1, characterized in that: The liquid raw materials include a mixture of carbon and nitrogen sources, trace elements, corn steep liquor, defoamer, wood ash, and nutrient solution.

3. The method according to claim 1, characterized in that: The carbon and nitrogen source mixture includes soybean meal, peanut meal powder, rice flour, and corn starch; the trace elements include yeast powder, potassium dihydrogen phosphate, and light calcium carbonate.

4. The method according to claim 3, characterized in that: The nutrient solution ingredients include brown sugar, corn steep liquor, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, sodium chloride, and calcium sulfate dihydrate.

5. The method according to claim 1, characterized in that: The self-generating nitrogen-fixing Bt used has nitrogen-fixing ability and biocontrol activity.

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