Plant perfume compound microorganism sustained release preparation as well as preparation method and application thereof

Abstract

The invention relates to the technical field of bacteriostatic agents, in particular to a plant perfume compound microorganism sustained release preparation as well as a preparation method and application thereof. The plant perfume compound microorganism sustained release preparation is prepared from the following raw materials in parts by mass: 2 to 4 parts of clove oil, 6 to 10 parts of citronellal, 1 part of an emulsifier, 2 to 4 parts of chitosan, 2 to 10 parts of sodium alginate, 2 to 6 parts of weathered coal humic acid, 2 to 6 parts of a compound bacterium agent, 10 to 20 parts of charcoal, 2 to 6 parts of amino-terminated polyamidoamine and 0.0002 to 0.0008 part of genipin. Through a triple synergistic mechanism of bacteriostasis of the complex microbial inoculant, slow release of plant active ingredients and rhizosphere micro-ecological regulation and control, the bottleneck that a single biotechnology is low in control effect and short in lasting effect is broken through, compared with chemical control, the cost is greatly reduced, and the method is suitable for an organic pineapple planting system and has remarkable economic and ecological benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial agents, and in particular to a plant-flavor composite microbial sustained-release preparation and a preparation method and application thereof. Background Art

[0002] Pineapple heart rot is a soil-borne disease that seriously harms pineapple production. The pathogen can infect pineapples throughout their growth period, mainly harming seedlings. In the early stage of the disease, the leaves are dark green and brown stripes appear at the base of the stem. The heart leaves are easy to pull up, and the whole plant dies in the later stage, seriously affecting the yield and quality.

[0003] Studies have shown that the disease is mainly caused by Phytophthora spp., including Phytophthora spp., Phytophthora spp., Phytophthora spp., etc. In terms of chemical control, although broad-spectrum fungicides have the advantage of high control efficiency, their long-term and large-scale use has led to multiple problems; the accumulation of pesticide residues in pineapple leaves and soil harms the ecological environment; the increasing drug resistance of pathogens has forced the increase of pesticide application, forming a vicious circle.

[0004] Recent studies have found that plant-derived ingredients such as clove oil and citronellal show good antibacterial potential. These two natural compounds have a significant inhibitory effect on soil-borne pathogens such as potato late blight and banana wilt by destroying the integrity of pathogen cell membranes and causing intracellular electrolyte exudation. As recognized safe natural extracts, their mature applications in spices, preservatives and other fields have laid the foundation for agricultural development.

[0005] In the development of new agents, compound preparations with differentiated action mechanisms have become a research hotspot. It is worth noting that although microbial agents are regarded as environmentally friendly alternatives, their antibacterial effects are easily affected by environmental factors such as temperature, pH value, and soil microbial communities. In actual applications, there is a prominent problem of insufficient stability, and poor compatibility with chemical agents, which can easily lead to an imbalance in the soil microbial community. Therefore, the development of preparations that have both stability, synergy, and ecological safety is a technical problem that needs to be solved urgently. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a plant flavor composite microbial sustained-release preparation and a preparation method and application thereof.

[0007] A plant flavor composite microbial sustained-release preparation, the raw materials of which include, by mass, 2-4 parts of clove oil, 6-10 parts of citronellal, 1 part of emulsifier, 2-4 parts of chitosan, 2-10 parts of sodium alginate, 2-6 parts of weathered coal humic acid, 2-6 parts of composite bacterial agent, 10-20 parts of biochar, 2-6 parts of amino-terminated polyamidoamine, and 0.0002-0.0008 parts of genipin.

[0008] Preferably, the eugenol content in clove oil is ≥85%.

[0009] Preferably, the emulsifier is Tween 80.

[0010] Preferably, the degree of deacetylation of chitosan is ≥95%, and the molecular weight is 40 - 50 kDa.

[0011] Preferably, the viable bacteria concentration in the compound microbial agent is ≥5×10 9 CFU / g.

[0012] Preferably, the compound microbial agent is obtained by fermenting a bagasse - rice bran matrix with Trichoderma harzianum TR - 7, Bacillus amyloliquefaciens BA - 9, and Pseudomonas fluorescens PF - 12.

[0013] Trichoderma harzianum TR - 7, Bacillus amyloliquefaciens BA - 9, and Pseudomonas fluorescens PF - 12 can all be freely purchased on the market.

[0014] Preferably, the compound microbial agent is prepared by the following steps: mixing Trichoderma harzianum TR - 7, Bacillus amyloliquefaciens BA - 9, and Pseudomonas fluorescens PF - 12 evenly to obtain a compound bacterial strain, using bagasse - rice bran as the matrix, and fermenting at 30 - 35°C for 90 - 100 h.

[0015] Preferably, in the compound bacterial strain, the viable bacteria concentration of Trichoderma harzianum TR - 7 is 0.5 - 1.5×10 8 CFU / g, the viable bacteria concentration of Bacillus amyloliquefaciens BA - 9 is 4 - 6×10 7 CFU / g, and the viable bacteria concentration of Pseudomonas fluorescens PF - 12 is 2.5 - 3.5×10 7 CFU / g.

[0016] Preferably, the water content of the bagasse - rice bran matrix is 40 - 50%, and the mass ratio of bagasse to rice bran is 1 - 2:1.

[0017] The preparation method of the above - mentioned plant - spice compound microbial sustained - release preparation includes the following steps: S1. Mix clove oil and citronellal, add an emulsifier, and perform ultrasonic emulsification at 40 - 50°C for 10 - 20 min to obtain a compound emulsion; S2. Sequentially add chitosan and sodium alginate to an acetic acid - sodium acetate buffer solution with pH = 5 - 5.5 and stir evenly. While stirring, drop the compound emulsion into it, perform high - pressure homogenization for 3 cycles, drop it into a calcium chloride aqueous solution pre - cooled to 5 - 10°C and stir for 15 - 20 min, drain, wash, add it to a sodium hydroxide solution and soak for 5 - 15 min, drain, wash, and freeze - dry to obtain a plant - derived bacteriostatic agent; S3. Add weathered coal humic acid to sodium hydroxide solution, stir at 50 - 60 °C for 1 - 2 h, centrifuge to obtain the supernatant, adjust the pH value of the system to 7 - 7.5, add compound bacterial agent and biochar thereto, perform ultrasonic treatment at 30 - 38 °C for 1 - 2 h, add plant - derived bacteriostatic agent and terminal amino polyamide amine, continue to stir for 10 - 20 min, dropwise add genipin solution thereto under stirring, stir at 25 - 30 °C for 1 - 2 h, centrifuge, wash, and freeze - dry.

[0018] Application of the above - mentioned plant spice compound microbial sustained - release preparation in the preparation of agricultural bacteriostatic agents.

[0019] Application of the above - mentioned plant spice compound microbial sustained - release preparation for bacteriostasis during the planting process of pineapple or ananas.

[0020] Beneficial effects The present invention uses Trichoderma harzianum TR - 7, Bacillus amyloliquefaciens BA - 9, and Pseudomonas fluorescens PF in combination, combines clove oil and citronellal to damage the cell membrane of pathogenic bacteria, realizes physical membrane rupture, enzymatic hydrolysis of cell walls, and nutritional competition, and synergistically inhibits the spore germination of pineapple heart rot pathogenic bacteria. The humic acid - biochar composite carrier can significantly improve the rhizosphere microenvironment, and the colonization density of beneficial bacteria is significantly increased.

[0021] The present invention emulsifies clove oil and citronellal, then forms a sodium alginate - Ca 2+ gel nucleus, and then realizes the transformation from nano - particles to a dense core - shell structure through alkalization treatment, effectively improving the stability of the active ingredients of clove oil and citronellal, reducing the loss of active ingredients; uses terminal amino polyamide amine to cross - link with chitosan and humic acid, prevents the aggregation of nano - particles by constructing steric hindrance, and can effectively avoid the direct contact between bacteria and plant - derived components, realizing the synergistic release of both.

[0022] The present invention adopts the synergistic effect of plant - derived active ingredients and compound bacterial agents, cooperates with the action of terminal amino polyamide amine and is solidified, greatly enhancing the bacteriostatic stability, which can delay the generation of pathogen drug resistance, significantly extend the bacteriostatic time limit, improve the control effect on pineapple heart rot, reduce the single - use dose of pesticides, reduce the control cost, and reduce pesticide residues.

[0023] The present invention breaks through the bottleneck of low efficacy and short duration of a single biological technology through the triple synergistic mechanisms of bacteriostasis by compound bacterial agents, slow release of plant active ingredients, and rhizosphere micro - ecological regulation. The cost is significantly reduced compared with chemical control, it is applicable to the organic pineapple planting system, and has significant economic and ecological benefits. Description of the drawings

[0024] Figure 1 It is a comparison chart of the bacteriostatic rates of the preparations obtained in Example 5 and Comparative Examples 1 - 2 against Phytophthora nicotianae.

[0025] Figure 2Incidence rate comparison chart of Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and carbendazim group.

[0026] Figure 3 Single fruit weight and sugar content of Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and carbendazim group.

[0027] Figure 4 Comparison chart of the root surface density of Pseudomonas fluorescens PF-12 in Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and carbendazim group. Detailed implementation manners

[0028] The present invention will be further explained below in conjunction with specific embodiments.

[0029] The clove oil used below has an eugenol content of ≥85%. The bagasse-rice bran is used as the matrix below, and the mass ratio of bagasse to rice bran is 3:2.

[0030] The Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 used below are all provided by a certain Nachuanglian Biotechnology Co., Ltd. in the mall.

[0031] Example 1 A plant spice composite microbial sustained-release preparation, the raw materials of which include: 10 g of clove oil, 30 g of citronellal, 5 g of Tween 80, 10 g of chitosan, 10 g of sodium alginate, 10 g of weathered coal humic acid, 10 g of a composite bacterial agent with a viable bacteria concentration of ≥5×10 9 CFU / g, 50 g of biochar, 10 g of terminal amino polyamide amine, and 0.001 g of genipin.

[0032] The composite bacterial agent is prepared by the following steps: Mix Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 evenly to obtain a composite bacterial strain (wherein, the viable bacteria concentration of Trichoderma harzianum TR-7 is 1×10 8 CFU / g, the viable bacteria concentration of Bacillus amyloliquefaciens BA-9 is 5×10 7 CFU / g, and the viable bacteria concentration of Pseudomonas fluorescens PF-12 is 3×10 7 CFU / g), and use bagasse-rice bran with a water content of 40% as the matrix, and ferment at a temperature of 30°C for 90 h.

[0033] The preparation method of the above plant spice composite microbial sustained-release preparation includes the following steps: S1. Mix clove oil and citronellal, add Tween 80, and perform ultrasonic emulsification at a temperature of 40°C for 10 min to obtain a composite emulsion; S2. Sequentially add chitosan and sodium alginate to 200 g of acetic acid - sodium acetate buffer solution with pH = 5 - 5.5, stir evenly, and dropwise add the composite emulsion under stirring. Treat it with a high - pressure homogenizer at 12000 r / min for 3 cycles. Drop the mixed solution into a calcium chloride aqueous solution with a mass fraction of 1% pre - cooled to 5℃ through a microfluidic device (flow rate 2 mL / min, pore diameter 200 μm), stir at a speed of 100 r / min for 15 min, drain, wash, add it to a sodium hydroxide solution with a concentration of 0.1 mol / L and soak for 5 min, drain, wash, and freeze - dry to obtain the plant - derived bacteriostatic agent; S3. Add weathered coal humic acid to 200 g of sodium hydroxide solution with a concentration of 0.1 mol / L, stir at 50℃ for 1 h, centrifuge to take the supernatant, adjust the pH value of the system to 7 - 7.5, add the composite bacterial agent and biochar, perform ultrasonic treatment at 30℃ for 1 h with an ultrasonic frequency of 30 kHz, add the plant - derived bacteriostatic agent and terminal amino polyamide - amine, continue to stir for 10 min, dropwise add 1 g of genipin solution with a mass fraction of 0.1% under stirring, stir at 25℃ for 1 h, centrifuge, wash, and freeze - dry.

[0034] Example 2 A plant - spice composite microbial sustained - release preparation, whose raw materials include: 20 g of clove oil, 50 g of citronellal, 5 g of Tween 80, 20 g of chitosan, 50 g of sodium alginate, 30 g of weathered coal humic acid, 30 g of a composite bacterial agent with a viable bacteria concentration ≥ 5×10 9 CFU / g, 100 g of biochar, 30 g of terminal amino polyamide - amine, and 0.004 g of genipin.

[0035] The composite bacterial agent is prepared by the following steps: Mix Trichoderma harzianum TR - 7, Bacillus amyloliquefaciens BA - 9, and Pseudomonas fluorescens PF - 12 evenly to obtain a composite bacterial strain (wherein, the viable bacteria concentration of Trichoderma harzianum TR - 7 is 1×10 8 CFU / g, the viable bacteria concentration of Bacillus amyloliquefaciens BA - 9 is 5×10 7 CFU / g, and the viable bacteria concentration of Pseudomonas fluorescens PF - 12 is 3×10 7 CFU / g), use bagasse - rice bran with a water content of 50% as the substrate, and ferment at 35℃ for 100 h.

[0036] The preparation method of the above - mentioned plant - spice composite microbial sustained - release preparation includes the following steps: S1. Mix clove oil and citronellal, add Tween 80, and perform ultrasonic emulsification at 50℃ for 20 min to obtain a composite emulsion; S2. Sequentially add chitosan and sodium alginate to 300 g of acetic acid - sodium acetate buffer solution with pH = 5 - 5.5, stir evenly, and while stirring, dropwise add the composite emulsion. Treat it with a high - pressure homogenizer at 12000 r / min for 3 cycles. Drop the mixed solution into a calcium chloride aqueous solution pre - cooled to 10 °C with a mass fraction of 2% through a microfluidic device (flow rate 2 mL / min, pore diameter 200 μm), stir at a speed of 200 r / min for 20 min, drain, wash, add it to a sodium hydroxide solution with a concentration of 0.2 mol / L, soak for 15 min, drain, wash, and freeze - dry to obtain a plant - derived bacteriostatic agent; S3. Add weathered coal humic acid to 400 g of sodium hydroxide solution with a concentration of 0.2 mol / L, stir at 60 °C for 2 h, centrifuge to take the supernatant, adjust the pH value of the system to 7 - 7.5, add the composite bacterial agent and biochar, perform ultrasonic treatment at 38 °C for 2 h with an ultrasonic frequency of 40 kHz, add the plant - derived bacteriostatic agent and terminal amino polyamide amine, continue to stir for 20 min, while stirring, dropwise add 2 g of genipin solution with a mass fraction of 0.2%, stir at 30 °C for 2 h, centrifuge, wash, and freeze - dry.

[0037] Example 3 A plant - spice composite microbial slow - release preparation, whose raw materials include: 12 g of clove oil, 45 g of citronellal, 5 g of Tween 80, 18 g of chitosan, 20 g of sodium alginate, 25 g of weathered coal humic acid, 15 g of a composite bacterial agent with a viable bacteria concentration ≥ 5×10 9 CFU / g, 90 g of biochar, 15 g of terminal amino polyamide amine, and 0.00204 g of genipin.

[0038] The composite bacterial agent is prepared by the following steps: Mix Trichoderma harzianum TR - 7, Bacillus amyloliquefaciens BA - 9, and Pseudomonas fluorescens PF - 12 evenly to obtain a composite bacterial strain (wherein, the viable bacteria concentration of Trichoderma harzianum TR - 7 is 1×10 8 CFU / g, the viable bacteria concentration of Bacillus amyloliquefaciens BA - 9 is 5×10 7 CFU / g, and the viable bacteria concentration of Pseudomonas fluorescens PF - 12 is 3×10 7 CFU / g), use bagasse - rice bran with a water content of 48% as the substrate, and ferment at 31 °C for 98 h.

[0039] The preparation method of the above - mentioned plant - spice composite microbial slow - release preparation includes the following steps: S1. Mix clove oil and citronellal, add Tween 80, and perform ultrasonic emulsification at 42 °C for 18 min to obtain a composite emulsion; S2. Sequentially add chitosan and sodium alginate to 220 g of acetic acid - sodium acetate buffer solution with a pH of 5 - 5.5, stir evenly, and while stirring, dropwise add the composite emulsion. Treat it with a high - pressure homogenizer at 12000 r / min for 3 cycles. Drop the mixed solution into an aqueous calcium chloride solution pre - cooled to 9 °C with a mass fraction of 1.3% through a microfluidic device (flow rate 2 mL / min, pore diameter 200 μm), stir at a speed of 180 r / min for 17 min, drain, wash, add it to a sodium hydroxide solution with a concentration of 0.17 mol / L and soak for 8 min, drain, wash, and freeze - dry to obtain the plant - derived antibacterial agent; S3. Add weathered coal humic acid to 350 g of sodium hydroxide solution with a concentration of 0.13 mol / L, stir at a temperature of 58 °C for 80 min, centrifuge to take the supernatant, adjust the pH value of the system to 7 - 7.5, add the composite bactericide and biochar, ultrasonically treat at a temperature of 36 °C for 80 min with an ultrasonic frequency of 39 kHz, add the plant - derived antibacterial agent and terminal amino polyamide amine, continue to stir for 12 min, while stirring, dropwise add 1.7 g of genipin solution with a mass fraction of 0.12%, stir at a temperature of 29 °C for 80 min, centrifuge, wash, and freeze - dry.

[0040] Example 4 A plant - spice composite microbial sustained - release preparation, whose raw materials include: 18 g of clove oil, 35 g of citronellal, 5 g of Tween 80, 12 g of chitosan, 40 g of sodium alginate, 15 g of weathered coal humic acid, 25 g of a composite bactericide with a viable bacteria concentration ≥ 5×10 9 CFU / g, 70 g of biochar, 25 g of terminal amino polyamide amine, and 0.00234 g of genipin.

[0041] The composite bactericide is prepared by the following steps: Mix Trichoderma harzianum TR - 7, Bacillus amyloliquefaciens BA - 9, and Pseudomonas fluorescens PF - 12 evenly to obtain a composite bacterial strain (wherein, the viable bacteria concentration of Trichoderma harzianum TR - 7 is 1×10 8 CFU / g, the viable bacteria concentration of Bacillus amyloliquefaciens BA - 9 is 5×10 7 CFU / g, and the viable bacteria concentration of Pseudomonas fluorescens PF - 12 is 3×10 7 CFU / g), use bagasse - rice bran with a water content of 42% as the substrate, and ferment at a temperature of 33 °C for 94 h.

[0042] The preparation method of the above - mentioned plant - spice composite microbial sustained - release preparation includes the following steps: S1. Mix clove oil and citronellal, add Tween 80, and ultrasonically emulsify at a temperature of 48 °C for 12 min to obtain a composite emulsion; S2. Add chitosan and sodium alginate to 280 g of acetic acid - sodium acetate buffer solution with a pH of 5 - 5.5 in sequence, stir evenly, and dropwise add the composite emulsion under stirring. Treat it with a high - pressure homogenizer at 12000 r / min for 3 cycles. Drop the mixed solution into an aqueous calcium chloride solution pre - cooled to 7 - 9 °C with a mass fraction of 1.3 - 1.7% through a microfluidic device (flow rate 2 mL / min, pore diameter 200 μm), stir at a speed of 120 r / min for 19 min, drain, wash, add it to a sodium hydroxide solution with a concentration of 0.13 mol / L and soak for 12 min, drain, wash, and freeze - dry to obtain the plant - derived bacteriostatic agent; S3. Add weathered coal humic acid to 250 g of sodium hydroxide solution with a concentration of 0.17 mol / L, stir at a temperature of 52 °C for 100 min, centrifuge to take the supernatant, adjust the pH value of the system to 7 - 7.5, add the composite bactericide and biochar, ultrasonically treat at a temperature of 32 °C for 100 min with an ultrasonic frequency of 33 kHz, add the plant - derived bacteriostatic agent and terminal amino polyamide amine, continue to stir for 18 min, dropwise add 1.3 g of genipin solution with a mass fraction of 0.18% under stirring, stir at a temperature of 27 °C for 100 min, centrifuge, wash, and freeze - dry.

[0043] Example 5 A plant - spice composite microbial sustained - release preparation, whose raw materials include: 15 g of clove oil, 40 g of citronellal, 5 g of Tween 80, 15 g of chitosan, 30 g of sodium alginate, 20 g of weathered coal humic acid, 20 g of a composite bactericide with a viable bacteria concentration ≥ 5×10 9 CFU / g, 80 g of biochar, 20 g of terminal amino polyamide amine, and 0.00225 g of genipin.

[0044] The composite bactericide is prepared by the following steps: Mix Trichoderma harzianum TR - 7, Bacillus amyloliquefaciens BA - 9, and Pseudomonas fluorescens PF - 12 evenly to obtain a composite bacterial strain (wherein, the viable bacteria concentration of Trichoderma harzianum TR - 7 is 1×10 8 CFU / g, the viable bacteria concentration of Bacillus amyloliquefaciens BA - 9 is 5×10 7 CFU / g, and the viable bacteria concentration of Pseudomonas fluorescens PF - 12 is 3×10 7 CFU / g), and use bagasse - rice bran with a water content of 45% as the substrate, and ferment at a temperature of 32 °C for 96 h.

[0045] The preparation method of the above - mentioned plant - spice composite microbial sustained - release preparation includes the following steps: S1. Mix clove oil and citronellal, add Tween 80, and ultrasonically emulsify at a temperature of 45 °C for 15 min to obtain a composite emulsion; S2. Sequentially add chitosan and sodium alginate to 250 g of acetic acid - sodium acetate buffer solution with pH = 5 - 5.5, stir evenly, and dropwise add the composite emulsion under stirring. Treat it with a high - pressure homogenizer at 12000 r / min for 3 cycles. Drop the mixed solution into a calcium chloride aqueous solution pre - cooled to 8 °C with a mass fraction of 1.5% through a microfluidic device (flow rate 2 mL / min, pore diameter 200 μm), stir at a speed of 150 r / min for 18 min, drain, wash, add it to a sodium hydroxide solution with a concentration of 0.15 mol / L and soak for 10 min, drain, wash, and freeze - dry to obtain the plant - derived bacteriostatic agent; S3. Add weathered coal humic acid to 300 g of sodium hydroxide solution with a concentration of 0.15 mol / L, stir at a temperature of 55 °C for 90 min, centrifuge to take the supernatant, adjust the pH value of the system to 7 - 7.5, add the composite bacterial agent and biochar, perform ultrasonic treatment at a temperature of 34 °C for 90 min with an ultrasonic frequency of 36 kHz, add the plant - derived bacteriostatic agent and terminal amino polyamide amine, continue to stir for 15 min, dropwise add 1.5 g of genipin solution with a mass fraction of 0.15% under stirring, stir at a temperature of 28 °C for 90 min, centrifuge, wash, and freeze - dry.

[0046] Comparative Example 1 A plant - spice composite microbial sustained - release preparation, whose raw materials include: 15 g of clove oil, 40 g of citronellal, 5 g of Tween 80, 15 g of chitosan, 30 g of sodium alginate, 20 g of weathered coal humic acid, 20 g of a composite bacterial agent with a viable bacteria concentration ≥ 5×10 9 CFU / g, 80 g of biochar, 20 g of terminal amino polyamide amine, and 0.00225 g of genipin.

[0047] The composite bacterial agent is prepared by the following steps: Mix Bacillus amyloliquefaciens BA - 9 and Pseudomonas fluorescens PF - 12 evenly to obtain a composite bacterial strain (wherein, the viable bacteria concentration of Bacillus amyloliquefaciens BA - 9 is 5×10 7 CFU / g, and the viable bacteria concentration of Pseudomonas fluorescens PF - 12 is 3×10 7 CFU / g), use bagasse - rice bran with a water content of 45% as the matrix, and ferment at a temperature of 32 °C for 96 h.

[0048] The preparation method of the above - mentioned plant - spice composite microbial sustained - release preparation includes the following steps: S1. Mix clove oil and citronellal, add Tween 80, and perform ultrasonic emulsification at a temperature of 45 °C for 15 min to obtain a composite emulsion; S2. Sequentially add chitosan and sodium alginate to 250 g of acetic acid - sodium acetate buffer solution with a pH of 5 - 5.5, stir evenly. While stirring, add the composite emulsion dropwise thereto, and process it through a high - pressure homogenizer at 12000 r / min for 3 cycles. Drop the mixed solution into an aqueous calcium chloride solution pre - cooled to 8 °C with a mass fraction of 1.5% at a flow rate of 2 mL / min through a microfluidic device (pore diameter 200 μm), stir at a speed of 150 r / min for 18 min, drain, wash, add it to a sodium hydroxide solution with a concentration of 0.15 mol / L and soak for 10 min, drain, wash, and freeze - dry to obtain a plant - derived bacteriostatic agent; S3. Add weathered coal humic acid to 300 g of sodium hydroxide solution with a concentration of 0.15 mol / L, stir at a temperature of 55 °C for 90 min, centrifuge to take the supernatant, adjust the pH value of the system to 7 - 7.5, add a composite bacterial agent and biochar thereto, perform ultrasonic treatment at a temperature of 34 °C for 90 min with an ultrasonic frequency of 36 kHz, add the plant - derived bacteriostatic agent and terminal amino polyamide, continue to stir for 15 min. While stirring, add 1.5 g of genipin solution with a mass fraction of 0.15% dropwise thereto, stir at a temperature of 28 °C for 90 min, centrifuge, wash, and freeze - dry.

[0049] Comparative Example 2 A plant - spice composite microbial sustained - release preparation, whose raw materials include: 15 g of clove oil, 40 g of citronellal, 5 g of Tween 80, 15 g of chitosan, 30 g of sodium alginate, 20 g of weathered coal humic acid, 20 g of a composite bacterial agent with a viable bacteria concentration ≥ 5×10 9 CFU / g, and 100 g of biochar.

[0050] The composite bacterial agent is prepared by the following steps: Mix Trichoderma harzianum TR - 7, Bacillus amyloliquefaciens BA - 9, and Pseudomonas fluorescens PF - 12 evenly to obtain a composite bacterial strain (wherein, the viable bacteria concentration of Trichoderma harzianum TR - 7 is 1×10 8 CFU / g, the viable bacteria concentration of Bacillus amyloliquefaciens BA - 9 is 5×10 7 CFU / g, and the viable bacteria concentration of Pseudomonas fluorescens PF - 12 is 3×10 7 CFU / g), use bagasse - rice bran with a water content of 45% as the substrate, and ferment at a temperature of 32 °C for 96 h.

[0051] The preparation method of the above - mentioned plant - spice composite microbial sustained - release preparation includes the following steps: S1. Mix clove oil and citronellal, add Tween 80, and perform ultrasonic emulsification at a temperature of 45 °C for 15 min to obtain a composite emulsion; S2. Sequentially add chitosan and sodium alginate to 250 g of acetic acid - sodium acetate buffer solution with pH = 5 - 5.5, stir evenly, and dropwise add the composite emulsion under stirring. Treat it with a high - pressure homogenizer at 12000 r / min for 3 cycles. Drop the mixed solution into an aqueous calcium chloride solution pre - cooled to 8 °C and with a mass fraction of 1.5% through a microfluidic device (flow rate 2 mL / min, pore diameter 200 μm), stir at a speed of 150 r / min for 18 min, drain, wash, add it to a sodium hydroxide solution with a concentration of 0.15 mol / L and soak for 10 min, drain, wash, and freeze - dry to obtain the plant - derived bacteriostatic agent; S3. Add weathered coal humic acid to 300 g of sodium hydroxide solution with a concentration of 0.15 mol / L, stir at a temperature of 55 °C for 90 min, centrifuge to take the supernatant, adjust the pH value of the system to 7 - 7.5, add the composite bactericide and biochar to it, perform ultrasonic treatment at a temperature of 34 °C for 90 min, the ultrasonic frequency is 36 kHz, add the plant - derived bacteriostatic agent and continue to stir for 15 min, centrifuge, wash, and freeze - dry.

[0052] Isolate the strain from the diseased fruits of pineapple heart rot in the pineapple planting area of Xuwen, Guangdong. After identification, it is Phytophthora nicotianae ( P. nicotianae ), and it is cultured using PDA medium. Take this Phytophthora nicotianae strain as the test strain.

[0053] Add the preparations obtained in Example 5 and Comparative Examples 1 - 2 to clear water (diluted 800 times) and disperse evenly to obtain the dispersion liquids of each group. Then, adopt the plate confrontation test. Place 4 Oxford cups at the bottom of the petri dish, and then pour the PDA medium into the petri dish. After the medium solidifies, take out the Oxford cups. And add 100 μL of the dispersion liquids of each group into the holes respectively; place a Phytophthora nicotianae mycelial block with the same size in the center of the solidified plate; for the blank control, only place a Phytophthora nicotianae mycelial block in the center of the PDA plate. Incubate at 25 °C. When the colony diameter of the blank control reaches more than 2 / 3 of the diameter of the petri dish, use a caliper to vertically measure the colony diameter of each group by the cross - method.

[0054] Bacteriostatic rate = (natural growth diameter of Phytophthora nicotianae - growth diameter of Phytophthora nicotianae inhibited by the dispersion liquid) ÷ natural growth diameter of Phytophthora nicotianae × 100%.

[0055] As Figure 1 shown, the bacteriostatic rate of the preparation obtained in Example 5 is the highest, superior to Comparative Examples 1 - 2 (P < 0.05).

[0056] In the pineapple planting area of Xuwen, Guangdong, plots with similar conditions were randomly divided into 5 groups (5 groups of examples, 1 group of comparative example 1, 1 group of comparative example 2, carbendazim group, blank group), with 5 replicates in each group, and each replicate had 20 vigorous, healthy winter honey pineapple seedlings with 6 leaves. Seven days before transplantation, the groups of examples 5, comparative example 1, and comparative example 2 were respectively applied with the preparation obtained in example 5 and the preparations obtained in comparative examples 1-2 (3 kg / mu) by mixing and applying in furrows with organic fertilizer (300 kg / mu), while the carbendazim group and the blank group were applied with biochar (3 kg / mu) and organic fertilizer (300 kg / mu) by mixing and applying in furrows.

[0057] Use a sterilized punch to prepare fungal blocks with a diameter of 5 mm and good growth. Inoculate all the seedlings at the base of the stem near the soil surface, make wounds in 4 directions with a sterilized pin, then cover with soil, and then irrigate.

[0058] After transplantation, the preparation obtained in example 5 and the preparations obtained in comparative examples 1-2 (both diluted 800 times) were diluted and dispersed with clear water, and then the above-mentioned diluted solutions were respectively sprayed on the leaves of the groups of examples 5, comparative example 1, and comparative example 2; the carbendazim group was sprayed with a 1000-fold diluted solution of carbendazim on the leaves, and the blank group was sprayed with an equal amount of clear water on the leaves. Spray once every 20 days.

[0059] During the entire test period, no fungicide was sprayed, and weeds were removed and pests were prevented in a timely manner manually. The disease incidence was counted.

[0060] Disease incidence = number of diseased plants ÷ total number of plants × 100%.

[0061] As Figure 2 shown, the disease incidence of the group of example 5 was the lowest, superior to the groups of comparative examples 1-2 and the carbendazim group (P < 0.05). And the disease incidence of the blank group was as high as over 90%, so it was not included in the Figure 2 display.

[0062] At the harvest stage, the pineapples obtained from each group were collected, weighed, and their sugar content was detected. As Figure 3 shown, the single fruit weight of the pineapples in the group of example 5 was the largest and the sugar content was the highest, both superior to the groups of comparative examples 1-2 and the carbendazim group (P < 0.05).

[0063] After harvesting, the soil on the roots of the pineapples in the groups of example 5, comparative example 1, comparative example 2, and carbendazim group was cleared, and then immersed in sterile water for 1 h, and the density of Pseudomonas fluorescens PF-12 on the root surface was calculated. As Figure 4 shown, the density of Pseudomonas fluorescens PF-12 on the root surface of the group of example 5 was the highest, superior to the groups of comparative examples 1-2 and the carbendazim group (P < 0.05).

[0064] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes should be covered within the protection scope of the present invention.

Claims

1. A plant flavor composite microbial sustained-release preparation, characterized in that: The raw materials include, by mass: 2-4 parts of clove oil, 6-10 parts of citronellal, 1 part of emulsifier, 2-4 parts of chitosan, 2-10 parts of sodium alginate, 2-6 parts of weathered coal humic acid, 2-6 parts of composite bacterial agent, 10-20 parts of biochar, 2-6 parts of amino-terminated polyamidoamine, and 0.0002-0.0008 parts of genipin; The composite bacterial agent is obtained by fermenting sugarcane bagasse-rice bran matrix with Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9 and Pseudomonas fluorescens PF-12.

2. The plant-flavor composite microbial sustained-release preparation according to claim 1, characterized in that: The eugenol content in clove oil is ≥85%, and the emulsifier is Tween 80.

3. The plant-flavor composite microbial sustained-release preparation according to claim 1, characterized in that: The deacetylation degree of chitosan is ≥95% and the molecular weight is 40-50kDa.

4. The plant-flavor composite microbial sustained-release preparation according to claim 1, characterized in that: The concentration of live bacteria in the compound bacterial agent is ≥5×10 9 CFU / g.

5. The plant-flavor composite microbial sustained-release preparation according to claim 1, characterized in that: The composite bacterial agent is prepared by the following steps: Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9 and Pseudomonas fluorescens PF-12 are uniformly mixed to obtain a composite bacterial strain, sugarcane bagasse-rice bran is used as a matrix, and fermented at 30-35°C for 90-100 hours.

6. The plant-flavor composite microbial sustained-release preparation according to claim 1, characterized in that: In the composite strain, the live bacterial concentration of Trichoderma harzianum TR-7 is 0.5-1.5×10 8 CFU / g, the live bacterial concentration of Bacillus amyloliquefaciens BA-9 is 4-6×10 7 CFU / g, the live bacterial concentration of Pseudomonas fluorescens PF-12 is 2.5-3.5×10 7 CFU / g.

7. A method for preparing the plant-flavor composite microbial sustained-release preparation according to any one of claims 1 to 6, characterized in that: The steps include: S1, mix clove oil and citronellal, add emulsifier, and ultrasonically emulsify at 40-50° C. for 10-20 min to obtain a composite emulsion; S2, add chitosan and sodium alginate to acetic acid-sodium acetate buffer with pH=5-5.5 in sequence and stir evenly, add the composite emulsion dropwise thereto under stirring, perform high pressure homogenization for 3 cycles, dropwise add to calcium chloride aqueous solution precooled to 5-10°C and stir for 15-20min, drain, wash, add to sodium hydroxide solution and soak for 5-15min, drain, wash, and freeze-dry to obtain a plant-derived antibacterial agent; S3. Add weathered coal humic acid to sodium hydroxide solution, stir at 50-60℃ for 1-2h, centrifuge to obtain supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 30-38℃ for 1-2h, add plant-derived antibacterial agent and amino-terminated polyamide amine, continue stirring for 10-20min, add genipin solution dropwise under stirring, stir at 25-30℃ for 1-2h, centrifuge, wash and freeze-dry.

8. Use of the plant-flavor composite microbial sustained-release preparation according to any one of claims 1 to 6 in the preparation of an agricultural antibacterial agent.

9. Use of the plant-flavor composite microbial sustained-release preparation according to any one of claims 1 to 6 for antibacterial use in the process of growing pineapples or pineapples.

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