Biochar fungicide for preventing and treating cotton verticillium wilt and preparation method thereof

Abstract

The invention provides a charcoal fungicide for preventing and treating cotton verticillium wilt and a preparation method of the charcoal fungicide, and belongs to the technical field of agricultural fungicide. According to the invention, the agricultural waste walnut green seedcase can be fully utilized to prepare the biochar material, and the biochar not only has good adsorption performance, but also is prepared through one-step pyrolysis, so that the process is simpler. The biochar is rich in functional groups, relatively high in specific surface area and relatively high in adsorption capacity, has a wide application prospect in the aspect of microbial agents, is good in prevention and treatment effect, and has a wide application value in the aspect of biological prevention and treatment of cotton verticillium wilt.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microbial agents, and in particular to a biochar microbial agent for preventing and treating cotton Verticillium wilt and a preparation method thereof. Background Art

[0002] Cotton Verticillium wilt is a soil-borne vascular fungal disease mainly caused by Verticillium dahliae and Verticillium wilt. The pathogen has a wide host range, multiple transmission pathways, and strong pathogenicity, which seriously affects the quality and yield of cotton. The disease is not easy to control, so it is called the "cancer" of cotton.

[0003] At present, the prevention and control of cotton Verticillium wilt is still mainly based on chemical pesticides, but excessive use of chemical pesticides will cause environmental pollution and pesticide residues, and pathogens will develop resistance, making the research and development of chemical pesticides more difficult. Biological control is environmentally friendly and can reduce agricultural production costs. It has the characteristics of being green, environmentally friendly, safe, low in resistance, renewable and sustainable. Therefore, the development of microbial agents is particularly important.

[0004] Bacillus subtilis has been used to prevent and control cotton Verticillium wilt, but a single Bacillus subtilis is greatly affected by the environment and has a low survival rate in the soil, which greatly affects the control effect. Biochar is a high-carbon solid residue formed by the pyrolysis of biomass raw materials under anaerobic or limited oxygen conditions. Biochar ash is rich in inorganic nutrients, which provide the necessary nutrient source for microorganisms, thereby driving the stability of microbial metabolism. This stability not only helps maintain the microbial population, but also promotes the health and balance of the soil ecosystem. Secondly, the unique pore structure and large specific surface area of ​​biochar also create an ideal habitat for microorganisms, enabling them to survive and reproduce stably in the soil. At the same time, the large specific surface area increases the contact area between microorganisms and biochar, further promoting their interaction.

[0005] In general, current research focuses on the application effect of biochar solidification microorganisms, while the preparation and optimization process of biochar agents are relatively lacking. This means that there are still some key links missing and deficiencies in the development and application of biochar agents, which require further research and exploration. In order to promote its wider application in cotton wilt, it is necessary to strengthen the screening research of biochar carriers, optimize the preparation process of biochar agents, and deeply explore its specific application effects in plant disease prevention and control. Summary of the invention

[0006] The purpose of the present invention is to provide a biochar agent for preventing and treating cotton Verticillium wilt and a preparation method thereof, thereby providing an effective prevention and treatment means for the crop disease.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a biochar agent, comprising the following steps:

[0009] Walnut green peel powder, KOH and urea are mixed and impregnated to obtain a pyrolysis raw material;

[0010] The pyrolysis raw material is subjected to pyrolysis treatment to obtain modified biochar;

[0011] The modified biochar is mixed with the bacterial solution and subjected to an oscillation adsorption treatment to obtain a biochar agent.

[0012] Preferably, the immersion treatment time is 2 to 4 hours.

[0013] Preferably, the temperature of the pyrolysis treatment is 750-850°C;

[0014] The pyrolysis treatment was carried out at N 2 Carried out in atmosphere.

[0015] Preferably, the bacterial liquid contains agricultural biocontrol strains.

[0016] Preferably, the agricultural biocontrol strain is selected from one or more of Bacillus Velez, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Paenibacillus polymyxa, Pseudomonas, radioactive soil Agrobacterium, Pasteurella, Bacillus pumilus, vine nitrogen-fixing bacteria or Bacillus cereus.

[0017] Preferably, the added ratio of the modified biochar is 0.02-0.03%.

[0018] Preferably, the OD of the bacterial solution is 600 0.5~2;

[0019] The diluent of the bacterial liquid is physiological saline;

[0020] The rotation speed of the oscillating adsorption treatment is 80 to 120 r / min;

[0021] The pH of the oscillation adsorption treatment is 4.5 to 6;

[0022] The temperature of the oscillation adsorption treatment is 25 to 35°C;

[0023] The time of the oscillation adsorption treatment is 2 to 5 hours.

[0024] Preferably, the bacterial liquid contains Bacillus subtilis 191-1, and the deposit number of the Bacillus subtilis 191-1 is CGMCC No.32923.

[0025] The present invention also provides a biochar agent prepared by the above preparation method.

[0026] The present invention also provides application of the biochar agent in preventing and controlling cotton Verticillium wilt.

[0027] Beneficial effects of the present invention:

[0028] 1. The present invention can make full use of agricultural waste walnut green peel to prepare biochar materials. The biochar not only has good adsorption performance, but also is prepared by one-step pyrolysis to make the process simpler. The biochar has rich functional groups, high specific surface area, strong adsorption capacity for Bacillus subtilis 191-1, and has broad application prospects in microbial agents.

[0029] 2. When the adsorption time was fixed at 3h, the temperature was fixed at 30℃, the biochar addition ratio was 0.025%, the pH was fixed at 5, and the speed was fixed at 100rpm, the adsorption capacity was the largest, which could reach 1.11×10 11 CFU / g. The number of effective live bacteria contained in the biochar agent prepared under this condition meets the national standards for agricultural microbial agents.

[0030] 3. The results of the greenhouse test showed that after adding biochar, the disease index of cotton Verticillium wilt dropped to 9.52, and the prevention efficiency could reach 88.52%. The prevention and control effect was good, and it has wide application value in the biological control of cotton Verticillium wilt. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1a This is a scanning electron micrograph of the modified biochar before adsorption and fixation of Bacillus;

[0032] Figure 1b This is a scanning electron micrograph of modified biochar after adsorption and fixation of Bacillus;

[0033] Figure 2 The nitrogen adsorption-desorption curve and pore size distribution diagram of the modified biochar;

[0034] Figure 3 is the adsorption amount at different fixed rotation speeds;

[0035] Figure 4 is the adsorption amount under different biochar addition ratios;

[0036] Figure 5 is the adsorption amount at different fixed pH values;

[0037] Figure 6 is the adsorption amount at different fixed temperatures;

[0038] Figure 7 Cotton phenotypes (from left to right: CK1, CK2, D1, D2);

[0039] Figure 8 This is a cross section of a cotton vascular tube (from left to right: CK1, D1, D2, CK2).

[0040] Biological Deposit Description

[0041] The Bacillus subtilis 191-1 involved in the present invention was deposited on December 5, 2024 in the General Microbiology Center of the China Microorganism Culture Collection Administration, referred to as CGMCC, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101, and the deposit number is CGMCC No. 32923. DETAILED DESCRIPTION

[0042] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1 Preparation of biochar agent

[0044] The collected waste walnut green peels were cleaned, dried, crushed and ground to obtain a yellow-brown powder of walnut green peel powder for later use. The walnut green peel powder, KOH and urea were mixed and immersed for 3 hours, dried and heated in N 2 Under atmosphere, high temperature pyrolysis was performed for 2 h, and after cooling, the modified biochar KNWP was obtained by washing and drying.

[0045] Take a bacterial liquid of Bacillus subtilis (strain: Bacillus subtilis 191-1) and centrifuge it at 5000 r / min for 10 min. Then wash the bacteria with 0.85% sterile saline. Repeat the centrifugation-washing operation 2-3 times and prepare a bacterial suspension with sterile saline.

[0046] The above bacterial suspension was diluted into bacterial suspensions of different concentrations and OD 600 The values ​​were compared with the standard curve of bacterial counts.

[0047] Take 0.1 g of the biochar prepared in the above step and put it into a conical flask, add 50 ml of OD 600 The bacterial suspension was adsorbed in a constant temperature shaking incubator. The oscillating adsorption conditions were: fixed speed of 100 r / min; biochar addition ratio of 0.025%, fixed pH 5, and fixed temperature of 30°C. After 3 hours, the supernatant was taken to measure its OD 600 Then the remaining mixture was passed through a 200-mesh sieve and washed 2-3 times with sterile water to obtain a biochar agent.

[0048] Experimental Example 1 Test of different fixed speeds in the preparation of biochar inoculants

[0049] 1. Preparation of modified biochar

[0050] The collected waste walnut green peels were cleaned, dried, crushed and ground to obtain a yellow-brown powder of walnut green peel powder for later use. The walnut green peel powder, KOH and urea were mixed and immersed for 3 hours, dried and heated in N 2 Under atmosphere, high temperature pyrolysis was performed for 2 h, and after cooling, the modified biochar KNWP was obtained by washing and drying.

[0051] 2. Preparation of biochar

[0052] (1) Take all the bacterial solution and centrifuge it at 5000 r / min for 10 min. Then wash the bacteria with 0.85% sterile saline. Repeat the centrifugation-washing operation 2-3 times to prepare a bacterial suspension with sterile saline.

[0053] (2) Dilute the above bacterial suspension into bacterial suspensions of different concentrations and calculate OD 600 The values ​​were compared with the standard curve of bacterial counts.

[0054] (3) Take 0.1 g of the biochar prepared in the above step and put it into a conical flask, add 50 ml of OD 600 The bacterial suspension was adsorbed in a constant temperature shaking incubator, and the supernatant was taken after 3 hours to determine its OD 600 Then the remaining mixture was passed through a 200-mesh sieve and washed 2-3 times with sterile water to obtain a biochar agent.

[0055] The oscillation adsorption conditions are as follows: fixed rotation speeds of 50 r / min, 100 r / min, 150 r / min, 200 r / min, and 250 r / min; the biochar addition ratio is 0.02%, the pH is fixed at 6.8, and the temperature is fixed at 30°C.

[0056] Experimental results and analysis

[0057] Biochar characterization results

[0058] Figure 1 is before adsorption ( Figure 1a )back( Figure 1b )SEM image of biochar, in which it can be seen that the prepared material shows a dense layer stacking of N-doped carbon material with an interconnected porous network, with some obvious pores, including mesopores and macropores. After adsorption, it can be clearly seen that the bacteria are adsorbed to the surface of the material and embedded in the pores.

[0059] Figure 2 It is the nitrogen adsorption-desorption curve and pore size distribution diagram of biochar. It can be seen that the biochar is mainly composed of microporous structure and has a large specific surface area, which is conducive to adsorption.

[0060] The table shows the specific surface area and pore size of modified biochar. It can be seen that the modified biochar has a larger specific surface area.

[0061] Figure 3 It can be seen that keeping other adsorption conditions unchanged and changing the fixed speed, the results show that when the speed is 100r / min, the adsorption amount is the highest, which can reach 2.07×10 10 CFU / g.

[0062] Experimental Example 2 Test of different biochar addition ratios in biochar agent preparation

[0063] 1. Preparation of modified biochar

[0064] The collected waste walnut green peels were cleaned, dried, crushed and ground to obtain a yellow-brown powder of walnut green peel powder for later use. The walnut green peel powder, KOH and urea were mixed and immersed for 3 hours, dried and heated in N 2 Under atmosphere, high temperature pyrolysis was performed for 2 h, and after cooling, the modified biochar KNWP was obtained by washing and drying.

[0065] 2. Preparation of biochar

[0066] (1) Take all the bacterial solution and centrifuge it at 5000 r / min for 10 min. Then wash the bacteria with 0.85% sterile saline. Repeat the centrifugation-washing operation 2-3 times to prepare a bacterial suspension with sterile saline.

[0067] (2) Dilute the above bacterial suspension into bacterial suspensions of different concentrations and calculate OD 600 The values ​​were compared with the standard curve of bacterial counts.

[0068] (3) Take 0.1 g of the biochar prepared in the above step and put it into a conical flask, add 50 ml of OD 600 The bacterial suspension was adsorbed in a constant temperature shaking incubator, and the supernatant was taken after 3 hours to determine its OD 600 Then the remaining mixture was passed through a 200-mesh sieve and washed 2-3 times with sterile water to obtain a biochar agent.

[0069] The oscillation adsorption conditions are as follows: biochar addition ratio: 0.0125%, 0.0250%, 0.0375%, 0.0500%, 0.0625%; fixed rotation speed of 100 r / min, solution pH of 6.8; fixed temperature of 30°C.

[0070] Among them, the number of live bacteria adsorbed by biochar was used as the evaluation index.

[0071] Experimental results and analysis

[0072] Figure 4It can be seen that keeping other adsorption conditions unchanged and changing the addition ratio of biochar, the results show that when the biochar addition ratio is 0.0250%, the adsorption amount is the highest, which can reach 1.11×10 11 CFU / g.

[0073] Experimental Example 3 Test of different fixed pH values ​​in the preparation of biochar agent

[0074] 1. Preparation of modified biochar

[0075] The collected waste walnut green peels were cleaned, dried, crushed and ground to obtain a yellow-brown powder of walnut green peel powder for later use. The walnut green peel powder, KOH and urea were mixed and immersed for 3 hours, dried and heated in N 2 Under atmosphere, high temperature pyrolysis was performed for 2 h, and after cooling, the modified biochar KNWP was obtained by washing and drying.

[0076] 2. Preparation of biochar

[0077] (1) Take all the bacterial solution and centrifuge it at 5000 r / min for 10 min. Then wash the bacteria with 0.85% sterile saline. Repeat the centrifugation-washing operation 2-3 times to prepare a bacterial suspension with sterile saline.

[0078] (2) Dilute the above bacterial suspension into bacterial suspensions of different concentrations and calculate OD 600 The values ​​were compared with the standard curve of bacterial counts.

[0079] (3) Take 0.1 g of the biochar prepared in the above step and put it into a conical flask, add 50 ml of OD 600 The bacterial suspension was adsorbed in a constant temperature shaking incubator, and the supernatant was taken after 3 hours to determine its OD 600 Then the remaining mixture was passed through a 200-mesh sieve and washed 2-3 times with sterile water to obtain a biochar agent.

[0080] The oscillating adsorption conditions are as follows: 1) fixed pH: 3, 5, 7, 9, 11; fixed rotation speed 100r / min; biochar addition ratio 0.0250%; fixed temperature 30°C.

[0081] Experimental results and analysis

[0082] from Figure 5 It can be seen that keeping other adsorption conditions unchanged and changing the fixed pH value, the results show that when the pH is 5, the adsorption amount is the highest, which can reach 8.20×10 10 CFU / g.

[0083] Experimental Example 4 Test of different fixed temperatures in the preparation of biochar inoculants

[0084] 1. Preparation of modified biochar

[0085] The collected waste walnut green peels were cleaned, dried, crushed and ground to obtain a yellow-brown powder of walnut green peel powder for later use. The walnut green peel powder, KOH and urea were mixed and immersed for 3 hours, dried and heated in N 2 Under atmosphere, high temperature pyrolysis was performed for 2 h, and after cooling, the modified biochar KNWP was obtained by washing and drying.

[0086] 2. Preparation of biochar

[0087] (1) Take all the bacterial solution and centrifuge it at 5000 r / min for 10 min. Then wash the bacteria with 0.85% sterile saline. Repeat the centrifugation-washing operation 2-3 times to prepare a bacterial suspension with sterile saline.

[0088] (2) Dilute the above bacterial suspension into bacterial suspensions of different concentrations and calculate OD 600 The values ​​were compared with the standard curve of bacterial counts.

[0089] (3) Take 0.1 g of the biochar prepared in the above step and put it into a conical flask, add 50 ml of OD 600 The bacterial suspension was adsorbed in a constant temperature shaking incubator, and the supernatant was taken after 3 hours to determine its OD 600 Then the remaining mixture was passed through a 200-mesh sieve and washed 2-3 times with sterile water to obtain a biochar agent.

[0090] The oscillating adsorption conditions are as follows: (1) fixed temperature: 25°C, 30°C, 35°C, 40°C, 45°C; fixed rotation speed of 100 r / min; biochar addition ratio of 0.0250%; fixed pH of 5.

[0091] Experimental results and analysis

[0092] from Figure 6 It can be seen that keeping other adsorption conditions unchanged and changing the fixed temperature does not change the adsorption amount significantly. Therefore, it can be considered that the effect of temperature on the adsorption effect is small. The embodiment provided by the present invention uses 30° C. as the fixed temperature.

[0093] Experimental Example 5 Study on the efficacy of biochar agent in preventing cotton Verticillium wilt

[0094] The biochar agent prepared in Example 1 was used for a prevention effect experiment.

[0095] Potted plant protection experiment

[0096] Four treatments were set up: CK1 (adding pathogen Verticillium dahliae V592), CK2 (blank group), D1 (pathogen + biochar agent), and D2 (pathogen + Bacillus subtilis ZC-214 suspension). Fifteen cotton seedlings were planted in each treatment, with three replicates.

[0097] When cotton had 2 to 4 true leaves, it was inoculated with Verticillium dahliae V592 for infection. The investigation began 15 days after inoculation, and the disease situation was investigated 15, 20, 25 and 30 days after inoculation. The disease situation was investigated according to the 5-level grading standard, and the disease index and disease prevention effect were calculated at the same time.

[0098] Disease index = ∑ (number of diseased plants at each level × value of the disease level) / (total number of plants surveyed × highest level value) × 100

[0099] Relative control effect = (control disease index - treatment disease index) / control disease index × 100;

[0100] Experimental results and analysis

[0101] Pot experiment results

[0102] The control effect of the present invention is shown in Table 1 below:

[0103] Table 1 Control effect

[0104] deal with Disease index Control effect (%) CK1 - - CK2 84.52 - D1 9.52 88.74 D2 21.83 74.17

[0105] Figure 7 These are the phenotypes of cotton under different treatments. From the figure, we can see that the cotton plants inoculated with Verticillium dahliae are shorter, wilt due to water loss, and in the most serious cases, the entire plant dies. However, the plants with the addition of the bacterial agent have dense leaves and grow better. There is no browning of the vascular tissue that has been opened, indicating that the bacterial agent has obvious preventive and control effects.

[0106] Figure 8 These are cross sections of the vascular bundles under different treatments. It can be seen that when the cotton stems inoculated with Verticillium dahliae alone were dissected, the vascular bundles turned brown, while the vascular bundles of the plants inoculated with the bacterial agent and bacterial solution were no different from those of the blank control group, and the plants grew normally.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a biochar agent, characterized in that: The following steps are involved: Walnut green peel powder, KOH and urea are mixed and impregnated to obtain a pyrolysis raw material; The pyrolysis raw material is subjected to pyrolysis treatment to obtain modified biochar; The modified biochar is mixed with the bacterial solution and subjected to an oscillation adsorption treatment to obtain a biochar agent.

2. The preparation method according to claim 1, characterized in that: The immersion treatment time is 2 to 4 hours.

3. The preparation method according to claim 1, characterized in that: The temperature of the pyrolysis treatment is 750-850°C; The pyrolysis treatment was carried out under a N2 atmosphere.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The bacterial liquid contains agricultural biocontrol strains.

5. The preparation method according to claim 4, characterized in that: The agricultural biocontrol strain is selected from one or more of Bacillus velez, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Paenibacillus polymyxa, Pseudomonas, radioactive soil Agrobacterium, Pasteurella, Bacillus pumilus, vine nitrogen-fixing bacteria or Bacillus cereus.

6. The preparation method according to claim 1, characterized in that: The added ratio of the modified biochar is 0.02-0.03%.

7. The preparation method according to claim 1, characterized in that: The OD of the bacterial solution 600 0.5~2; The diluent of the bacterial liquid is physiological saline; The rotation speed of the oscillating adsorption treatment is 80 to 120 r / min; The pH of the oscillation adsorption treatment is 4.5 to 6; The temperature of the oscillation adsorption treatment is 25 to 35°C; The time of the oscillation adsorption treatment is 2 to 5 hours.

8. The preparation method according to claim 1, characterized in that: The bacterial liquid contains Bacillus subtilis 191-1, and the preservation number of the Bacillus subtilis 191-1 is CGMCC No.32923.

9. The biochar agent prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the biochar agent according to claim 9 in preventing and controlling cotton Verticillium wilt.

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