Method and application of livestock and poultry biochar loaded with Trichoderma

Abstract

The present invention provides a method and application for loading Trichoderma onto livestock and poultry biochar. The Trichoderma is fermented into a seed liquid through liquid fermentation, which is then subjected to shallow solid-state fermentation to obtain activated Trichoderma. Trichoderma microcapsules are simultaneously prepared from zinc sub-nanowires, sodium alginate, nutrient solution microspheres, and Trichoderma seed liquid. The livestock and poultry biochar is simultaneously loaded with the activated Trichoderma and the Trichoderma microcapsules to prepare charcoal-based Trichoderma. The present invention belongs to the technical field of microbial fertilizers. The Trichoderma seed liquid contained in the Trichoderma microcapsules can be further activated by using a nutrient solution. At the same time, the volatile organic compounds produced by the metabolism of the activated Trichoderma can form a gel network structure with the zinc sub-nanowires through multi-level interactions, ensuring that the activation of the Trichoderma seed liquid is not affected. This facilitates further replenishment when the activated Trichoderma finishes functioning, and continues to maintain the high-activity Trichoderma for continuous functioning. The method has the advantages of long-term and high efficiency, and provides good technical support for the preparation of charcoal-based Trichoderma.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fertilizers, and particularly relates to a method and application of loading Trichoderma on livestock and poultry biochar. Background Art

[0002] Trichoderma is a widely distributed fungus in nature, belonging to the subphylum Deuteromycotina, class Hypomycetes, and order Combretales. As a ubiquitous fungus in nature, it is often found in rotten wood, dead branches, fallen leaves, and soil. Common Trichoderma fungi include T. reesei, T. viride, T. harzianum, T. acanthosporum, and T. longibrachiatum. T. reesei and T. viride can be used to produce cellulase and laccase, promoting the production of fuel ethanol and biodiesel and the industrial development and utilization of straw biomass. T. harzianum, T. acanthosporum, and T. longibrachiatum can be used as biological control agents and soil conditioners to control plant fungal and pest diseases, as well as soil metal pollution, and have important ecological and application value.

[0003] Existing research shows that microbial fertilizers are highly efficient, harmless, and environmentally friendly, effectively promoting plant growth and improving the soil environment. Trichoderma, as a highly effective antagonistic microorganism, possesses properties such as improving the soil environment, promoting plant growth, and being environmentally friendly, making it an ideal candidate for microbial fertilizer. However, when applied alone, Trichoderma spores have a short survival period and are easily inactivated, resulting in a reduction in the number of viable spores during actual use, thus affecting their germination, growth, and colonization capabilities. Furthermore, the application methods for Trichoderma are relatively complex, such as mixing with soil, spreading, or spraying. This is not only labor-intensive but also susceptible to soil antibacterial factors and pesticide residues, further reducing its effectiveness.

[0004] Prior art uses a loading system to encapsulate Trichoderma to enhance its effectiveness. However, most existing studies have used activated Trichoderma mixed with the loading system to create a Trichoderma-encapsulated structure, while few studies have simultaneously encapsulated unactivated Trichoderma and highly active Trichoderma. Therefore, research on methods for simultaneously encapsulating both Trichoderma and highly active Trichoderma could extend the duration of Trichoderma's active application and achieve long-term efficacy. Summary of the Invention

[0005] Technical problems to be solved: In response to the above technical problems, the purpose of the present invention is to provide a method and application of loading Trichoderma on livestock and poultry biochar, wherein Trichoderma is fermented into a seed liquid through liquid fermentation, and then activated Trichoderma is obtained through shallow solid-state fermentation. At the same time, Trichoderma microcapsules are prepared from zinc sub-nanowires, sodium alginate, nutrient solution microspheres and Trichoderma seed liquid. The livestock and poultry biochar is simultaneously loaded with activated Trichoderma and Trichoderma microcapsules to prepare charcoal-based Trichoderma, which belongs to the technical field of microbial fertilizers. The Trichoderma seed liquid contained in the Trichoderma microcapsules in the present invention can be further activated by the nutrient solution. At the same time, the volatile organic compounds produced by the metabolism of the activated Trichoderma can form a gel network structure with the zinc sub-nanowires through multi-level interactions, ensuring that the activation of the Trichoderma seed liquid is not affected, facilitating further replenishment when the activated Trichoderma finishes taking effect, and continuing to maintain the high-activity Trichoderma to continue to take effect. It has the advantages of long-term and high efficiency, and provides good technical support for the preparation of charcoal-based Trichoderma.

[0006] Technical solution: A method for loading Trichoderma on livestock and poultry biochar, comprising the following steps:

[0007] S1. Trichoderma liquid fermentation to produce Trichoderma seed liquid, and then shallow solid-state fermentation to produce activated Trichoderma;

[0008] S2. Using zinc sub-nanowires and sodium alginate to load Trichoderma seed solution and nutrient solution microspheres to prepare Trichoderma microcapsules;

[0009] S3. The livestock and poultry biochar, activated Trichoderma and Trichoderma microcapsules are uniformly mixed to prepare a charcoal-based Trichoderma.

[0010] Furthermore, the Trichoderma in step S1 is one or more of Trichoderma koningii, Trichoderma viride, Trichoderma harzianum, Trichoderma acanthosporum, and Trichoderma longibrachiatum.

[0011] Furthermore, the conditions for liquid fermentation in step S1 are fermentation temperature of 25-30° C., fermentation time of 1-3 days, and rotation speed of 100-200 r / min.

[0012] Furthermore, the conditions for the shallow tray solid-state fermentation in step S1 are as follows: an inoculation amount of 5-15%, a culture temperature of 25-30° C., and a culture time of 3-5 days.

[0013] Furthermore, the preparation method of the Trichoderma microcapsules in step S2 is as follows: ① zinc chloride and phosphomolybdic acid are uniformly dissolved in a ratio of 1: (1-4), and then oleic acid and oleylamine are added and stirred at 40-80 ° C for 2-6 hours, washed and centrifuged to obtain zinc sub-nanowires; ② zinc sub-nanowires and sodium alginate are dissolved in ethanol in a ratio of (1-5): 1 to prepare a wall material solution, and then nutrient solution microspheres and Trichoderma seed solution are mixed in a ratio of (1-3): 1 to prepare a core material solution; ③ the wall material solution and the core material solution are mixed in a ratio of 1: (2-4) and dropped into a calcium chloride solution with a concentration of 2-4%, washed and filtered to prepare Trichoderma microcapsules.

[0014] Furthermore, the preparation method of the nutrient solution microspheres in step S2 is: uniformly mixing the nutrient solution and the chitosan solution at a ratio of 1: (2.5-5) and dripping into a sodium hydroxide solution, and filtering and washing to obtain the nutrient solution microspheres.

[0015] Furthermore, the composition of the nutrient solution is glucose 2.0-4.0 g / L, peptone 1.0-2.0 g / L, yeast extract 0.5-1.0 g / L, mannitol 0.5-1.5 g / L, potassium dihydrogen phosphate 0.3-0.6 g / L, calcium carbonate 0.2-0.6 g / L, and eucommia leaf extract 0.5-2.0 g / L.

[0016] Furthermore, in step S3, the ratio of livestock and poultry biochar, activated Trichoderma and Trichoderma microcapsules is (10-20):(1-4):(1-3).

[0017] A carbon-based Trichoderma prepared by any of the methods described above.

[0018] Furthermore, the carbon-based Trichoderma is used in the preparation of microbial organic fertilizer.

[0019] Beneficial effects:

[0020] 1. In the present invention, zinc sub-nanowires and sodium alginate are combined with each other through hydrogen bonding to form a network structure, which loads nutrient solution microspheres and Trichoderma seed solution to complete the embedding of unactivated Trichoderma, and then loads them into livestock and poultry biochar with activated Trichoderma to achieve simultaneous embedding of unactivated Trichoderma and activated Trichoderma, which can increase the loading capacity of Trichoderma.

[0021] 2. The livestock and poultry biochar-loaded activated Trichoderma in the present invention can metabolize and produce volatile organic compounds (such as alkanes, alkenes, esters, etc.) when it works. The zinc sub-nanowires adsorb volatile organic compounds and form a self-supporting elastic organic gel network through the multi-level interaction formed between zinc ions and polyacid clusters. It is attached to the outer layer of the sodium alginate structure, ensuring that the activated Trichoderma and the unactivated Trichoderma do not affect each other, so that the Trichoderma in the microcapsule can better complete the activation process.

[0022] 3. The livestock and poultry biochar-loaded Trichoderma microcapsules of the present invention contain Trichoderma seed liquid and nutrient solution microspheres. When the activated Trichoderma begins to take effect, the Trichoderma seed liquid in the Trichoderma microcapsules enters the activation stage, decomposes the chitosan outer layer of the nutrient solution microspheres and releases the nutrient solution, providing the nutrients required for the activation of Trichoderma. At the same time, on the one hand, the Eucommia leaf extract can promote the growth and reproduction of Trichoderma, shorten the time used in the delay phase and accelerate the entry into the logarithmic phase of reproduction, so that the concentration of Trichoderma in the Trichoderma microcapsules increases rapidly. On the other hand, Trichoderma successively decomposes the sodium alginate and zinc sub-nanowires in the microcapsule structure, which can separate zinc ions and molybdenum elements, which can further activate the activity of the Trichoderma metabolic enzyme system and promote the rapid growth and thickening of the fungal mycelium, thereby shortening the time used for Trichoderma activation and facilitating timely release and continued efficient function.

[0023] 4. The method of loading Trichoderma on livestock and poultry biochar in the present invention can simultaneously load highly active Trichoderma and unactivated Trichoderma. The effects of the two-stage Trichoderma are efficient and long-lasting, which broadens new research ideas for the preparation of charcoal-based Trichoderma. It can not only improve the utilization efficiency of Trichoderma in soil remediation, plant control, fertilizer preparation and other fields, but also has good development prospects. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with examples, which are provided to explain the present invention and are not limited to the following examples:

[0025] Example 1

[0026] A method for loading Trichoderma on livestock and poultry biochar comprises the following steps:

[0027] S1. Ferment Trichoderma harzianum at 28°C and 120 rpm for 2 days to produce a T. harzianum seed solution. 10 mL of the T. harzianum seed solution was then inoculated into solid culture medium and fermented in a shallow tray at 28°C for 4 days to produce activated T. harzianum.

[0028] S2. Weigh 4g glucose, 1g peptone, 0.75g yeast extract, 1g mannitol, 0.3g potassium dihydrogen phosphate, 0.4g calcium carbonate, 2g eucommia leaf extract to prepare 1L nutrient solution; 50mL nutrient solution and 150mL chitosan solution were mixed and dropped into sodium hydroxide solution, filtered and washed to obtain nutrient solution microspheres;

[0029] S3. Preparation of Trichoderma harzianum Microcapsules

[0030] ① Dissolve 10g zinc chloride and 40g phosphomolybdic acid in octadecene reagent, then add oleic acid and oleylamine and stir at 60°C for 4h. Wash repeatedly with cyclohexane and ethanol and centrifuge three times to obtain zinc sub-nanowires.

[0031] ② Dissolve 40g of zinc sub-nanowires and 20g of sodium alginate in ethanol to prepare a wall material solution, and then mix 20g of nutrient solution microspheres and 20g of Trichoderma harzianum seed liquid to prepare a core material solution;

[0032] ③ Add 20g of wall material solution and 40g of core material solution dropwise into 3% calcium chloride solution, wash and filter to prepare Trichoderma microcapsules.

[0033] S4. 100 g of livestock and poultry biochar, 10 g of activated Trichoderma harzianum and 20 g of Trichoderma harzianum microcapsules were mixed evenly to prepare a charcoal-based Trichoderma harzianum.

[0034] Example 2

[0035] A method for loading Trichoderma on livestock and poultry biochar comprises the following steps:

[0036] S1. Trichoderma aspergillus was fermented at 25°C and 160 rpm for 2 days to prepare a Trichoderma aspergillus seed solution, and then 10 mL of the Trichoderma aspergillus seed solution was inoculated into a solid culture medium. The solid culture medium was fermented in a shallow dish at 25°C for 4 days to prepare activated Trichoderma aspergillus;

[0037] S2. Weigh 4g glucose, 1g peptone, 0.75g yeast extract, 1g mannitol, 0.3g potassium dihydrogen phosphate, 0.4g calcium carbonate, 2g eucommia leaf extract to prepare 1L nutrient solution; 50mL nutrient solution and 150mL chitosan solution were mixed and dropped into sodium hydroxide solution, filtered and washed to obtain nutrient solution microspheres;

[0038] S3. Preparation of Trichoderma aspergillus microcapsules

[0039] ① Dissolve 10g zinc chloride and 40g phosphomolybdic acid in octadecene reagent, then add oleic acid and oleylamine and stir at 60°C for 4h. Wash repeatedly with cyclohexane and ethanol and centrifuge three times to obtain zinc sub-nanowires.

[0040] ② Dissolve 40g of zinc sub-nanowires and 20g of sodium alginate in ethanol to prepare a wall material solution, and then mix 20g of nutrient solution microspheres and 20g of Trichoderma aspergillus seed liquid to prepare a core material solution;

[0041] ③ Add 20g of wall material solution and 40g of core material solution dropwise into 3% calcium chloride solution, wash and filter to prepare Trichoderma microcapsules.

[0042] S4. 100 g of livestock and poultry biochar, 10 g of activated Trichoderma aspergillus and 20 g of Trichoderma aspergillus microcapsules were mixed evenly to prepare charcoal-based Trichoderma aspergillus.

[0043] Example 3

[0044] A method for loading Trichoderma on livestock and poultry biochar comprises the following steps:

[0045] S1. Ferment Trichoderma harzianum at 28°C and 120 rpm for 2 days to produce a T. harzianum seed solution. 10 mL of the T. harzianum seed solution was then inoculated into solid culture medium and fermented in a shallow tray at 28°C for 4 days to produce activated T. harzianum.

[0046] S2. Trichoderma aspergillus was fermented at 25 ° C and 160 r / min for 2 days to prepare Trichoderma aspergillus seed solution, and then 10 mL of Trichoderma aspergillus seed solution was inoculated into solid culture medium, and solid fermentation was carried out in a shallow dish at 25 ° C for 4 days to prepare activated Trichoderma aspergillus, and then 50 g of activated Trichoderma harzianum and 50 g of activated Trichoderma aspergillus were mixed to prepare activated composite Trichoderma;

[0047] S3. Weigh 4g glucose, 1g peptone, 0.75g yeast extract, 1g mannitol, 0.3g potassium dihydrogen phosphate, 0.4g calcium carbonate, 2g eucommia leaf extract to prepare 1L nutrient solution; 50mL nutrient solution and 150mL chitosan solution were mixed and dropped into sodium hydroxide solution, filtered and washed to obtain nutrient solution microspheres;

[0048] S4. Preparation of composite Trichoderma microcapsules

[0049] ① Dissolve 10g zinc chloride and 40g phosphomolybdic acid in octadecene reagent, then add oleic acid and oleylamine and stir at 60°C for 4h. Wash repeatedly with cyclohexane and ethanol and centrifuge three times to obtain zinc sub-nanowires.

[0050] ② Evenly mix 20 mL of Trichoderma harzianum seed solution and 20 mL of Trichoderma acanthosporum seed solution to prepare a composite Trichoderma seed solution;

[0051] ③ Dissolve 40g of zinc sub-nanowires and 20g of sodium alginate in ethanol to prepare a wall material solution, and then mix 20g of nutrient solution microspheres and 20g of composite Trichoderma seed liquid to prepare a core material solution;

[0052] ④ Add 20g of wall material solution and 40g of core material solution dropwise into 3% calcium chloride solution, wash and filter to prepare Trichoderma microcapsules.

[0053] S5. 100 g of livestock and poultry biochar, 10 g of activated composite Trichoderma and 20 g of composite Trichoderma microcapsules were mixed evenly to prepare a carbon-based composite Trichoderma.

[0054] Example 4

[0055] A method for loading Trichoderma on livestock and poultry biochar comprises the following steps:

[0056] S1. Ferment Trichoderma harzianum at 28°C and 120 rpm for 2 days to produce a seed solution of Trichoderma harzianum. 5 mL of the seed solution was then inoculated into solid culture medium and fermented in a shallow tray at 28°C for 4 days to produce activated Trichoderma harzianum.

[0057] S2. Trichoderma aspergillus was fermented at 25 ° C and 120 r / min for 2 days to prepare Trichoderma aspergillus seed solution, and then 5 mL of Trichoderma aspergillus seed solution was inoculated into solid culture medium, and solid fermentation was carried out in a shallow dish at 25 ° C for 4 days to prepare activated Trichoderma aspergillus, and then 50 g of activated Trichoderma harzianum and 50 g of activated Trichoderma aspergillus were mixed to prepare activated composite Trichoderma;

[0058] S3. Weigh 2g glucose, 1g peptone, 0.75g yeast extract, 1g mannitol, 0.5g potassium dihydrogen phosphate, 0.4g calcium carbonate, 2g eucommia leaf extract to prepare 1L nutrient solution; 30mL nutrient solution and 120mL chitosan solution were mixed and dropped into sodium hydroxide solution, filtered and washed to obtain nutrient solution microspheres;

[0059] S4. Preparation of composite Trichoderma microcapsules

[0060] ① Dissolve 10g zinc chloride and 40g phosphomolybdic acid in octadecene reagent, then add oleic acid and oleylamine and stir at 60°C for 4h. Wash repeatedly with cyclohexane and ethanol and centrifuge three times to obtain zinc sub-nanowires.

[0061] ② Evenly mix 20 mL of Trichoderma harzianum seed solution and 20 mL of Trichoderma acanthosporum seed solution to prepare a composite Trichoderma seed solution;

[0062] ③ Dissolve 40g of zinc sub-nanowires and 20g of sodium alginate in ethanol to prepare a wall material solution, and then mix 20g of nutrient solution microspheres and 20g of composite Trichoderma seed liquid to prepare a core material solution;

[0063] ④ Add 20g of wall material solution and 40g of core material solution dropwise into 3% calcium chloride solution, wash and filter to prepare Trichoderma microcapsules.

[0064] S5. 100 g of livestock and poultry biochar, 10 g of activated composite Trichoderma and 20 g of composite Trichoderma microcapsules were mixed evenly to prepare a carbon-based composite Trichoderma.

[0065] Example 5

[0066] A method for loading Trichoderma on livestock and poultry biochar comprises the following steps:

[0067] S1. Ferment Trichoderma harzianum at 28°C and 120 rpm for 2 days to produce a T. harzianum seed solution. 10 mL of the T. harzianum seed solution was then inoculated into solid culture medium and fermented in a shallow tray at 28°C for 4 days to produce activated T. harzianum.

[0068] S2. Trichoderma aspergillus was fermented at 25 ° C and 160 r / min for 2 days to prepare Trichoderma aspergillus seed solution, and then 10 mL of Trichoderma aspergillus seed solution was inoculated into solid culture medium, and solid fermentation was carried out in a shallow dish at 25 ° C for 4 days to prepare activated Trichoderma aspergillus, and then 50 g of activated Trichoderma harzianum and 50 g of activated Trichoderma aspergillus were mixed to prepare activated composite Trichoderma;

[0069] S3. Weigh 4g glucose, 1g peptone, 0.75g yeast extract, 1g mannitol, 0.3g potassium dihydrogen phosphate, 0.4g calcium carbonate, 2g eucommia leaf extract to prepare 1L nutrient solution; 50mL nutrient solution and 150mL chitosan solution were mixed and dropped into sodium hydroxide solution, filtered and washed to obtain nutrient solution microspheres;

[0070] S4. Preparation of composite Trichoderma microcapsules

[0071] ① Dissolve 10g zinc chloride and 30g phosphomolybdic acid in octadecene reagent, then add oleic acid and oleylamine and stir at 60°C for 6h. Wash repeatedly with cyclohexane and ethanol and centrifuge three times to obtain zinc sub-nanowires.

[0072] ② Evenly mix 20 mL of Trichoderma harzianum seed solution and 20 mL of Trichoderma acanthosporum seed solution to prepare a composite Trichoderma seed solution;

[0073] ③ Dissolve 40g of zinc sub-nanowires and 20g of sodium alginate in ethanol to prepare a wall material solution, and then mix 20g of nutrient solution microspheres and 20g of composite Trichoderma seed liquid to prepare a core material solution;

[0074] ④ Add 20g of wall material solution and 40g of core material solution dropwise into 3% calcium chloride solution, wash and filter to prepare Trichoderma microcapsules.

[0075] S5. 100 g of livestock and poultry biochar, 15 g of activated composite Trichoderma and 20 g of composite Trichoderma microcapsules were mixed evenly to prepare a carbon-based composite Trichoderma.

[0076] Example 6

[0077] A method for loading Trichoderma on livestock and poultry biochar comprises the following steps:

[0078] S1. Ferment Trichoderma harzianum at 28°C and 120 rpm for 2 days to produce a T. harzianum seed solution. 10 mL of the T. harzianum seed solution was then inoculated into solid culture medium and fermented in a shallow tray at 28°C for 4 days to produce activated T. harzianum.

[0079] S2. Trichoderma aspergillus was fermented at 25 ° C and 160 r / min for 2 days to prepare Trichoderma aspergillus seed solution, and then 10 mL of Trichoderma aspergillus seed solution was inoculated into solid culture medium, and solid fermentation was carried out in a shallow dish at 25 ° C for 4 days to prepare activated Trichoderma aspergillus, and then 50 g of activated Trichoderma harzianum and 50 g of activated Trichoderma aspergillus were mixed to prepare activated composite Trichoderma;

[0080] S3. Weigh 4g glucose, 1g peptone, 0.75g yeast extract, 1g mannitol, 0.3g potassium dihydrogen phosphate, 0.4g calcium carbonate, 2g eucommia leaf extract to prepare 1L nutrient solution; 50mL nutrient solution and 150mL chitosan solution were mixed and dropped into sodium hydroxide solution, filtered and washed to obtain nutrient solution microspheres;

[0081] S4. Preparation of composite Trichoderma microcapsules

[0082] ① Dissolve 10g zinc chloride and 40g phosphomolybdic acid in octadecene reagent, then add oleic acid and oleylamine and stir at 60°C for 4h. Wash repeatedly with cyclohexane and ethanol and centrifuge three times to obtain zinc sub-nanowires.

[0083] ② Evenly mix 40 mL of Trichoderma harzianum seed solution and 20 mL of Trichoderma acanthosporum seed solution to prepare a composite Trichoderma seed solution;

[0084] ③ Dissolve 40g of zinc sub-nanowires and 10g of sodium alginate in ethanol to prepare a wall material solution, and then mix 40g of nutrient solution microspheres and 20g of composite Trichoderma seed liquid to prepare a core material solution;

[0085] ④ Add 20g of wall material solution and 40g of core material solution dropwise into 3% calcium chloride solution, wash and filter to prepare Trichoderma microcapsules.

[0086] S5. 100 g of livestock and poultry biochar, 10 g of activated composite Trichoderma and 20 g of composite Trichoderma microcapsules were mixed evenly to prepare a carbon-based composite Trichoderma.

[0087] Example 7

[0088] A method for loading Trichoderma on livestock and poultry biochar comprises the following steps:

[0089] S1. Ferment Trichoderma harzianum at 28°C and 120 rpm for 2 days to produce a T. harzianum seed solution. 10 mL of the T. harzianum seed solution was then inoculated into solid culture medium and fermented in a shallow tray at 28°C for 4 days to produce activated T. harzianum.

[0090] S2. Trichoderma aspergillus was fermented at 25 ° C and 160 r / min for 2 days to prepare Trichoderma aspergillus seed solution, and then 10 mL of Trichoderma aspergillus seed solution was inoculated into solid culture medium, and solid fermentation was carried out in a shallow dish at 25 ° C for 4 days to prepare activated Trichoderma aspergillus, and then 50 g of activated Trichoderma harzianum and 50 g of activated Trichoderma aspergillus were mixed to prepare activated composite Trichoderma;

[0091] S3. Weigh 2g glucose, 2g peptone, 1g yeast extract, 1.5g mannitol, 0.5g potassium dihydrogen phosphate, 0.5g calcium carbonate, 2g eucommia leaf extract to prepare 1L nutrient solution; 50mL nutrient solution and 150mL chitosan solution were mixed and dropped into sodium hydroxide solution, filtered and washed to obtain nutrient solution microspheres;

[0092] S4. Preparation of composite Trichoderma microcapsules

[0093] ① Dissolve 10g zinc chloride and 40g phosphomolybdic acid in octadecene reagent, then add oleic acid and oleylamine and stir at 60°C for 4h. Wash repeatedly with cyclohexane and ethanol and centrifuge three times to obtain zinc sub-nanowires.

[0094] ② Evenly mix 20 mL of Trichoderma harzianum seed solution and 20 mL of Trichoderma acanthosporum seed solution to prepare a composite Trichoderma seed solution;

[0095] ③ Dissolve 40g of zinc sub-nanowires and 20g of sodium alginate in ethanol to prepare a wall material solution, and then mix 20g of nutrient solution microspheres and 20g of composite Trichoderma seed liquid to prepare a core material solution;

[0096] ④ Add 20g of wall material solution and 40g of core material solution dropwise into 3% calcium chloride solution, wash and filter to prepare Trichoderma microcapsules.

[0097] S5. 100 g of livestock and poultry biochar, 15 g of activated composite Trichoderma and 20 g of composite Trichoderma microcapsules were mixed evenly to prepare a carbon-based composite Trichoderma.

[0098] Comparative Example 1

[0099] The difference between this comparative example and Example 3 is that zinc sub-nanowires are not added.

[0100] A method for loading Trichoderma on livestock and poultry biochar comprises the following steps:

[0101] S1. Ferment Trichoderma harzianum at 28°C and 120 rpm for 2 days to produce a T. harzianum seed solution. 10 mL of the T. harzianum seed solution was then inoculated into solid culture medium and fermented in a shallow tray at 28°C for 4 days to produce activated T. harzianum.

[0102] S2. Trichoderma aspergillus was fermented at 25 ° C and 160 r / min for 2 days to prepare Trichoderma aspergillus seed solution, and then 10 mL of Trichoderma aspergillus seed solution was inoculated into solid culture medium, and solid fermentation was carried out in a shallow dish at 25 ° C for 4 days to prepare activated Trichoderma aspergillus, and then 50 g of activated Trichoderma harzianum and 50 g of activated Trichoderma aspergillus were mixed to prepare activated composite Trichoderma;

[0103] S3. Weigh 4g glucose, 1g peptone, 0.75g yeast extract, 1g mannitol, 0.3g potassium dihydrogen phosphate, 0.4g calcium carbonate, 2g eucommia leaf extract to prepare 1L nutrient solution; 50mL nutrient solution and 150mL chitosan solution were mixed and dropped into sodium hydroxide solution, filtered and washed to obtain nutrient solution microspheres;

[0104] S4. Preparation of composite Trichoderma microcapsules

[0105] ① Evenly mix 20 mL of Trichoderma harzianum seed solution and 20 mL of Trichoderma acanthosporum seed solution to prepare a composite Trichoderma seed solution;

[0106] ② Dissolve 20g of sodium alginate in ethanol to prepare a wall material solution, and then mix 20g of nutrient solution microspheres and 20g of composite Trichoderma seed liquid to prepare a core material solution;

[0107] ③ Add 20g of wall material solution and 40g of core material solution dropwise into 3% calcium chloride solution, wash and filter to prepare Trichoderma microcapsules.

[0108] S5. 100 g of livestock and poultry biochar, 10 g of activated composite Trichoderma and 20 g of composite Trichoderma microcapsules were mixed evenly to prepare a carbon-based composite Trichoderma.

[0109] Comparative Example 2

[0110] The difference between this comparative example and Example 3 is that no Eucommia leaf extract was added.

[0111] A method for loading Trichoderma on livestock and poultry biochar comprises the following steps:

[0112] S1. Ferment Trichoderma harzianum at 28°C and 120 rpm for 2 days to produce a T. harzianum seed solution. 10 mL of the T. harzianum seed solution was then inoculated into solid culture medium and fermented in a shallow tray at 28°C for 4 days to produce activated T. harzianum.

[0113] S2. Trichoderma aspergillus was fermented at 25 ° C and 160 r / min for 2 days to prepare Trichoderma aspergillus seed solution, and then 10 mL of Trichoderma aspergillus seed solution was inoculated into solid culture medium, and solid fermentation was carried out in a shallow dish at 25 ° C for 4 days to prepare activated Trichoderma aspergillus, and then 50 g of activated Trichoderma harzianum and 50 g of activated Trichoderma aspergillus were mixed to prepare activated composite Trichoderma;

[0114] S3 weighed 4g glucose, 1g peptone, 0.75g yeast extract, 1g mannitol, 0.3g potassium dihydrogen phosphate, 0.4g calcium carbonate, prepared into 1L nutrient solution; 50mL nutrient solution and 150mL chitosan solution were mixed and dropped into sodium hydroxide solution, filtered and washed to obtain nutrient solution microspheres;

[0115] S4. Preparation of composite Trichoderma microcapsules

[0116] ① Dissolve 10g zinc chloride and 40g phosphomolybdic acid in octadecene reagent, then add oleic acid and oleylamine and stir at 60°C for 4h. Wash repeatedly with cyclohexane and ethanol and centrifuge three times to obtain zinc sub-nanowires.

[0117] ② Evenly mix 20 mL of Trichoderma harzianum seed solution and 20 mL of Trichoderma acanthosporum seed solution to prepare a composite Trichoderma seed solution;

[0118] ③ Dissolve 40g of zinc sub-nanowires and 20g of sodium alginate in ethanol to prepare a wall material solution, and then mix 20g of nutrient solution microspheres and 20g of composite Trichoderma seed liquid to prepare a core material solution;

[0119] ④ Add 20g of wall material solution and 40g of core material solution dropwise into 3% calcium chloride solution, wash and filter to prepare Trichoderma microcapsules.

[0120] S5. 100 g of livestock and poultry biochar, 10 g of activated composite Trichoderma and 20 g of composite Trichoderma microcapsules were mixed evenly to prepare a carbon-based composite Trichoderma.

[0121] Comparative Example 3

[0122] The difference between this comparative example and Example 3 is that no composite Trichoderma microcapsules are added.

[0123] A method for loading Trichoderma on livestock and poultry biochar comprises the following steps:

[0124] S1. Ferment Trichoderma harzianum at 28°C and 120 rpm for 2 days to produce a T. harzianum seed solution. 10 mL of the T. harzianum seed solution was then inoculated into solid culture medium and fermented in a shallow tray at 28°C for 4 days to produce activated T. harzianum.

[0125] S2. Trichoderma aspergillus was fermented at 25 ° C and 160 r / min for 2 days to prepare Trichoderma aspergillus seed solution, and then 10 mL of Trichoderma aspergillus seed solution was inoculated into solid culture medium, and solid fermentation was carried out in a shallow dish at 25 ° C for 4 days to prepare activated Trichoderma aspergillus, and then 50 g of activated Trichoderma harzianum and 50 g of activated Trichoderma aspergillus were mixed to prepare activated composite Trichoderma;

[0126] S3. Evenly mix 100 g of livestock and poultry biochar and 10 g of activated composite Trichoderma to prepare a carbon-based composite Trichoderma.

[0127] Performance evaluation

[0128] (1) Effective viable bacteria count

[0129] The effective viable bacteria count of the carbon-based Trichoderma prepared in Examples 1-7 and Comparative Examples 1-3 was determined as follows: 5.0 g of the sample was placed in a sterilized triangular flask containing 45 mL of sterile water, placed in a shaker and shaken at 28 ° C and 170 rpm for 30 min, then taken out, 0.1 mL of the sample solution was taken, 0.9 mL of sterile water was added, and the mixture was shaken for 3 seconds to prepare a 10-fold diluted bacterial suspension, and so on until the bacterial suspension was gradiently diluted 10 times. 6 times, take 0.1 mL of bacterial suspension and spread it on Trichoderma selective culture medium, place it in a constant temperature incubator at 28℃ for 72 hours, then take it out and calculate the effective viable bacteria count.

[0130] Table 1 Effective viable bacteria counts of Examples 1-7 and Comparative Examples 1-3

[0131] <![CDATA[Number of viable bacteria (×10 8 CFU / g)]]> <![CDATA[Number of viable bacteria (×10 8 CFU / g)]]> Example 1 2.46 Example 6 2.13 Example 2 2.28 Example 7 2.25 Example 3 2.32 Comparative Example 1 2.10 Example 4 2.20 Comparative Example 2 2.05 Example 5 2.16 Comparative Example 3 1.85

[0132] As can be seen from Table 1, the effective viable counts of charcoal-based Trichoderma prepared in Examples 1-7 are all higher than those in Comparative Examples 1-3, while Comparative Example 1 does not add zinc sub-nanowires, Comparative Example 2 does not add eucommia leaf extract, and Comparative Example 3 does not add Trichoderma microcapsules. The results show that the molybdenum element in the zinc sub-nanowires and the nutrients contained in the eucommia leaf extract can promote the growth of Trichoderma during the determination of the effective viable count and increase the effective viable count of Trichoderma.

[0133] (2) Seedling cultivation test

[0134] The seedling raising effects of Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 were evaluated. Five treatments were set, namely, a blank group, an Example 3 group, a Comparative Example 1 group, a Comparative Example 2 group and a Comparative Example 3 group. The seedling raising test was carried out as follows:

[0135] ① Seed pretreatment: 50 plump and uniform watermelon seeds were selected for each treatment and sterilized in 55°C warm water for 20 minutes with constant stirring. When the temperature dropped to 28°C, the seeds were placed in a constant temperature incubator at 28°C. After soaking for 6 hours, the seeds were taken out and the mucus on the surface of the seeds was washed off. Then, two pieces of filter paper with a diameter of 12.5 cm were moistened with distilled water and spread on the bottom of the culture dish. The seeds were spread flat in the culture dish, and an appropriate amount of distilled water was added to the dish and placed in a constant temperature incubator. Water was replenished every day according to the actual situation. Sowing was carried out when the germination rate reached 90%.

[0136] ② Seedling treatment: 1.5 kg of seedling matrix was weighed for each treatment, and the charcoal-based Trichoderma prepared in Example 3 and Comparative Examples 1-3 and the seedling matrix were mixed well. The mixed matrix was placed in a hole tray and stamped with a stamping plate to a depth of about 1 cm. The seed buds were uniformly sown in the holes with the downward direction and covered with soil and watered. After sowing, the emergence time and the emergence rate were recorded; at the same time, when the seedlings had 3 leaves and 1 heart, samples were taken to measure the plant height, stem diameter, leaf area, and seedling index.

[0137] Table 2 Seedling emergence rate (%) of blank group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group and Comparative Example 3 group

[0138] Seedling emergence time (days) Blank group Example 3 group Comparative Example 1 Comparative Example 2 Comparative Example 3 1 0.00 0.00 0.00 0.00 0.00 2 4.02 14.51 8.36 7.21 5.17 3 20.12 60.58 50.16 44.01 30.23 4 35.24 95.17 75.41 68.59 47.33 5 75.42 100.00 96.23 90.32 78.15 6 90.03 - 100.00 100.00 92.96 7 92.76 - - - 100.00 8 96.32 - - - - 9 100.00 - - - -

[0139] Table 3 Growth of watermelon seedlings in the blank group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group and Comparative Example 3 group

[0140] Growth evaluation indicators Plant height (cm) Stem diameter (mm) <![CDATA[Leaf area (cm 2 )]]> Seedling index Blank group 7.33 3.20 19.53 0.17 Example 3 group 9.57 4.18 28.62 0.35 Comparative Example 1 8.63 3.77 25.37 0.28 Comparative Example 2 8.58 3.58 24.62 0.25 Comparative Example 3 8.21 3.42 22.05 0.22

[0141] As shown in Table 2, compared with the blank group, the germination rate of watermelon seeds in Example 3 reached 100% on the 5th day, while the germination rates of Comparative Examples 1, 2 and 3 reached 100% on the 6th and 7th days respectively, indicating that the application of charcoal-based Trichoderma can promote the germination of watermelon seeds; and as shown in Table 3, the plant height, stem thickness, leaf area and seedling index of Example 3 are all better than those of Comparative Examples 1-3, indicating that the activated Trichoderma embedded in livestock and poultry biochar can play a growth-promoting role on watermelon seeds in advance, and the Trichoderma seed liquid in the Trichoderma microcapsules loaded by livestock and poultry biochar decomposes the chitosan in the outer layer of the nutrient solution microspheres to release nutrients, and the Trichoderma seed liquid uses the nutrients to complete the activation stage, and the eucommia leaf extract contained in the nutrient solution can promote The activation of Trichoderma seed liquid shortens the activation time; in addition, the volatile organic compounds produced by the initial activated Trichoderma can form a gel network structure through multi-level interactions with the zinc sub-nanowires on the outer layer of the Trichoderma microcapsule, and adhere to the outermost layer of the microcapsule, ensuring that the activation process of the Trichoderma seed liquid is not disturbed. Therefore, when the metabolic activity of the activated Trichoderma that took effect first weakens, the activated Trichoderma seed liquid in the Trichoderma microcapsule is completely metabolically active and can be further released, achieving the effect of long-term and efficient promotion of watermelon seed germination, shortening the watermelon seed germination time, thereby increasing the seedling rate, and greatly improving the root and stem growth effect of the watermelon seedlings, thereby effectively increasing the plant height, stem thickness and leaf area of ​​the watermelon seedlings, achieving the effect of strengthening the seedlings.

[0142] (3) Experiment on the prevention and treatment of melon root rot by carbon-based Trichoderma

[0143] The control effect of the carbon-based Trichoderma prepared in Example 3 and Comparative Examples 1-3 on melon root rot was analyzed. The specific test method is as follows:

[0144] ① Inoculation of Fusarium verticillioides: Use a 7mm puncher to punch the outermost surface of the activated Fusarium verticillioides. Then transfer three pieces of the cake to PD medium and culture on a shaker set at 25°C and 120 rpm for 7 days. Set aside and adjust the pathogen spore concentration to 1×10 6 After sowing the melon seeds, the pathogen was inoculated into the soil at a volume of 5 mL per seedling. ② Melon seed pretreatment: Rinse the melon seeds with clean water, then soak them for about 2 hours, place them in a constant temperature box at 25°C for germination, and start sowing after the seeds germinate.

[0145] ③ Prevention and control test design: The test was divided into 5 groups, namely blank group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group and Comparative Example 3 group. At the same time, 5 melon seeds were sown in each group. Then, after the blank control seedlings emerged, the number of melon root rot diseased plants in each group was recorded to calculate the prevention and control effect.

[0146] Table 4 Control effect of Example 3 and Comparative Examples 1-3 on melon root rot

[0147] Control effect (%) Blank group 0.00 Example 3 group 95.61 Comparative Example 1 80.25 Comparative Example 2 78.35 Comparative Example 3 70.25

[0148] As can be seen from Table 4, the charcoal-based Trichoderma prepared in Example 3 has a significantly better control effect on melon root rot than comparative examples 1-3, with a control effect of more than 90%. The results show that the simultaneous encapsulation of activated Trichoderma and Trichoderma seed liquid in livestock and poultry biochar can release highly active Trichoderma in two steps to act on Fusarium verticillioides, thereby inhibiting the harmful effects of Fusarium verticillioides on melon rhizomes. In contrast, in comparative example 1, no zinc sub-nanowires were added, which would result in the simultaneous release of Trichoderma seed liquid and activated Trichoderma embedded in the Trichoderma microcapsules. The blending of Trichoderma seed liquid and activated Trichoderma would affect the overall inhibitory effect of Trichoderma, thereby reducing the inhibitory effect on Fusarium verticillioides. In comparative example 2, no eucommia leaf extract was added, which would result in the Trichoderma seed liquid in the Trichoderma microcapsules not completing the activation stage after the activated Trichoderma had finished taking effect, resulting in the subsequent release of Trichoderma activity not reaching the best state, thereby reducing its control effect on melon root rot. In general, the livestock and poultry biochar loaded activated Trichoderma and Trichoderma microcapsules in the embodiment can realize the two-stage release of highly active Trichoderma, ensuring that Trichoderma has a good control effect on plant pathogens and achieving the purpose of efficient control.

[0149] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for loading Trichoderma on livestock and poultry biochar, characterized in that: The following steps are involved: S1 Trichoderma liquid fermentation to produce Trichoderma seed liquid, and then shallow solid-state fermentation to produce activated Trichoderma; the Trichoderma is Trichoderma harzianum, Trichoderma spinulosa one or more; S2. Using zinc sub-nanowires and sodium alginate to load Trichoderma seed solution and nutrient solution microspheres to prepare Trichoderma microcapsules; The preparation method of the Trichoderma microcapsules comprises the following steps: ① dissolving zinc chloride and phosphomolybdic acid in a ratio of 1: (1-4) uniformly, then adding oleic acid and oleylamine, stirring and reacting at 40-80° C. for 2-6 hours, washing and centrifuging to obtain zinc sub-nanowires; ② dissolving zinc sub-nanowires and sodium alginate in ethanol in a ratio of (1-5): 1 to prepare a wall material solution, then mixing nutrient solution microspheres and Trichoderma seed solution in a ratio of (1-3): 1 to prepare a core material solution; ③ mixing the wall material solution and the core material solution in a ratio of 1: (2-4) and dripping them into a calcium chloride solution with a concentration of 2-4%, washing and filtering to prepare the Trichoderma microcapsules; The preparation method of the nutrient solution microspheres comprises: uniformly mixing a nutrient solution and a chitosan solution at a ratio of 1: (2.5-5), dripping the mixture into a sodium hydroxide solution, and filtering and washing the mixture to obtain the nutrient solution microspheres; the nutrient solution comprises 2.0-4.0 g / L of glucose, 1.0-2.0 g / L of peptone, 0.5-1.0 g / L of yeast extract, 0.5-1.5 g / L of mannitol, 0.3-0.6 g / L of potassium dihydrogen phosphate, 0.2-0.6 g / L of calcium carbonate, and 0.5-2.0 g / L of eucommia leaf extract; S3. The livestock and poultry biochar, activated Trichoderma and Trichoderma microcapsules are uniformly mixed to prepare a charcoal-based Trichoderma fertilizer.

2. The method for loading Trichoderma on livestock and poultry biochar according to claim 1, characterized in that: The conditions for liquid fermentation in step S1 are fermentation temperature of 25-30° C., fermentation time of 1-3 days, and rotation speed of 100-200 r / min.

3. The method for loading Trichoderma on livestock and poultry biochar according to claim 1, wherein: The conditions for the shallow tray solid-state fermentation in step S1 are as follows: an inoculation amount of 5-15%, a culture temperature of 25-30° C., and a culture time of 3-5 days.

4. The method for loading Trichoderma on livestock and poultry biochar according to claim 1, characterized in that: In step S3, the ratio of livestock and poultry biochar, activated Trichoderma and Trichoderma microcapsules is (10-20): (1-4): (1-3).

5. A charcoal-based Trichoderma fertilizer prepared according to the method according to any one of claims 1 to 4.

6. Application of the charcoal-based Trichoderma fertilizer according to claim 5 in the preparation of microbial organic fertilizer.