Compound preparation of compound bacterial agent compounded plant source bacteriostatic agent as well as preparation method and application of compound preparation

Abstract

The invention relates to the technical field of bacteriostatic agents, in particular to a compound bacterial agent compounded plant source bacteriostatic agent compound preparation as well as a preparation method and application thereof. The composite microbial agent compounded plant source bacteriostatic agent composite preparation is prepared from the following raw materials in parts by mass: 70 to 220 parts of plant source bacteriostatic agent, 50 to 100 parts of fermentation product, 10 to 30 parts of carboxyl-terminated polyamidoamine, 40 to 60 parts of activated nano montmorillonite, 1 part of azo initiator, 10 to 30 parts of binder and 1 to 10 parts of silane coupling agent, the plant-derived bacteriostatic agent is prepared by the following steps: adding chitosan into an acetic acid-sodium acetate buffer solution, uniformly mixing, adding sodium alginate, uniformly stirring, adding plant-derived bioactive components, uniformly stirring, dropwise adding into a calcium chloride solution, curing, and drying in vacuum; the fermentation product is obtained by fermenting the bagasse-soybean meal substrate with a complex microbial inoculant; the composite microbial agent is prepared from trichoderma harzianum T-22, bacillus subtilis BS-5 and pseudomonas fluorescens PF-3.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteriostatic agents, and particularly to a compound preparation of a compound bacterium agent and a plant-derived bacteriostatic agent, and its preparation method and application. Background Art

[0002] Pineapple is a tropical fruit, originally from the tropical regions of South America, and is now widely cultivated in tropical and subtropical regions around the world. It is rich in nutrients such as vitamin C, vitamin B1, and potassium, which are beneficial to physical health. Appropriate consumption of pineapple can provide rich nutrition and promote physical health. Field pineapple refers to the pineapple planted in the fields, usually referring to the pineapple planted on a large scale in farmland.

[0003] Currently, the large-scale cultivation of pineapple faces severe challenges in disease prevention and control. Wilt disease, as a typical systemic infection disease, is caused by the pineapple wilt virus and is characterized by a fast transmission speed and a deep degree of harm. The disease shows typical symptoms such as root browning and necrosis, leaf yellowing and curling, and fruit development deformity. In severe cases, it can cause yield losses in the entire planting area and is listed as one of the most threatening phytosanitary diseases in the pineapple industry.

[0004] Wilt disease generally causes disease by interfering with plant hormone metabolism and nutrient transport pathways. Infected plants show progressive wilting characteristics. Initially, only the new leaves turn yellow, and irreversible damages such as abnormal proliferation of aerial roots and browning of the main root xylem gradually appear as the disease progresses. The prevalence of the disease is closely related to the soil pathogen load, cultivation management mode, and plant resistance level. Continuous cropping plots are prone to form disease outbreak areas due to pathogen accumulation.

[0005] There are significant technical bottlenecks in the existing disease prevention and control system. As a quinoline-based protective low-toxic fungicide, copper quinolate can form a tight protective film on the surface of field pineapple after spraying, has a strong affinity with plants, and is resistant to rain erosion. However, the long-term continuous use of pesticides alone is prone to risks of resistance and increased dosage, which will increase the control cost and environmental pressure. The field colonization rate of traditional biocontrol bacteria (such as Trichoderma) is low, and the control effect on soil-borne diseases is unstable. Benzimidazole fungicides cause soil microbial imbalance, with a high mutation rate of pathogen drug resistance. At the same time, directly spraying plant active substances is easily photolyzed and inactivated, and the effective period is insufficient. Summary of the Invention

[0006] The purpose of the present invention is to solve the disadvantages existing in the prior art, and to provide a compound preparation of a compound bacterium agent and a plant-derived bacteriostatic agent, and its preparation method and application.

[0007] A compound preparation of a compound bacterial agent and a plant-derived bacteriostatic agent, the raw materials of which include, by mass: 70-220 parts of a plant-derived bacteriostatic agent, 50-100 parts of a fermentation product, 10-30 parts of a terminal carboxyl polyamide amine, 40-60 parts of activated nano-montmorillonite, 1 part of an azo initiator, 10-30 parts of a binder, and 1-10 parts of a silane coupling agent.

[0008] The plant-derived bacteriostatic agent is prepared by the following steps: adding chitosan into an acetic acid-sodium acetate buffer solution and mixing evenly, adding sodium alginate and stirring evenly, adding a plant-derived bioactive ingredient and stirring evenly, dropping into a calcium chloride solution, curing, and vacuum drying.

[0009] The fermentation product is obtained by fermenting a bagasse-soybean meal substrate with a compound bacterial agent; the compound bacterial agent includes: Trichoderma harzianum T-22, Bacillus subtilis BS-5, and Pseudomonas fluorescens PF-3; the carbon-nitrogen ratio in the bagasse-soybean meal substrate is 24-26:1.

[0010] Trichoderma harzianum T-22, Bacillus subtilis BS-5, and Pseudomonas fluorescens PF-3 can all be freely purchased on the market.

[0011] Preferably, the mass ratio of the plant-derived bioactive ingredient to chitosan and sodium alginate is 5-15:1:1-5.

[0012] Preferably, the plant-derived bioactive ingredient is prepared by the following specific steps: drying and pulverizing aloe vera leaves and sophora flavescens roots respectively, then mixing, and performing the first reflux extraction with water to obtain medicinal residues a and an extract a , concentrating the extract a to obtain a thick paste a ; pulverizing the medicinal residues a and performing the second reflux extraction with an ethanol aqueous solution to obtain an extract b , concentrating the extract b to obtain a thick paste b ; mixing the thick paste a and the thick paste b evenly.

[0013] More preferably, the mass ratio of dried and pulverized aloe vera leaves to dried and pulverized sophora flavescens roots is 10:1-4.

[0014] More preferably, during the first reflux extraction, the mass ratio of the material to the liquid is 1:5-10, the reflux extraction temperature is 95-100 °C, and the reflux extraction time is 5-10 h.

[0015] More preferably, during the second reflux extraction, the mass fraction of the ethanol aqueous solution is 90-95%, the mass ratio of the material to the liquid is 1:4-6, the reflux extraction temperature is 75-80 °C, and the reflux extraction time is 5-15 h.

[0016] More preferably, the thick paste a has a relative density of 1.1 - 1.3, and the thick paste b has a relative density of 1.4 - 1.6.

[0017] Preferably, the mass fraction of the calcium chloride solution is 1 - 2%.

[0018] Preferably, the curing temperature is 5 - 10 °C, and the curing time is 10 - 30 min.

[0019] Preferably, in the compound microbial agent, the viable count of Trichoderma harzianum T - 22 is 1 - 2×10 8 CFU / g, the viable count of Bacillus subtilis BS - 5 is 1 - 5×10 7 CFU / g, and the viable count of Pseudomonas fluorescens PF - 3 is 1 - 3×10 7 CFU / g.

[0020] Preferably, the fermentation temperature is 25 - 30 °C, and the fermentation time is 70 - 80 h.

[0021] Preferably, the activated nano - montmorillonite is obtained by activating nano - montmorillonite at 120 - 130 °C for 1 - 2 h.

[0022] Preferably, the azo initiator is azodiisobutyramidine hydrochloride.

[0023] Preferably, the binder is polyvinyl alcohol.

[0024] Preferably, the silane coupling agent is silane coupling agent KH - 550.

[0025] The preparation method of the above - mentioned compound microbial agent compounded with a plant - derived bacteriostatic agent compound preparation comprises the following steps: adding terminal - carboxyl polyamidoamine to the citric acid / EDTA mixed solution, stirring evenly, adding activated nano - montmorillonite, and performing ultrasonic treatment for 10 - 30 min, adjusting the pH value of the system to 7.2 - 7.6, adding an azo initiator, continuing ultrasonic treatment at 50 - 60 °C for 10 - 30 min, cooling to 30 - 40 °C, adding the fermentation product, the plant - derived bacteriostatic agent, the binder, and the silane coupling agent, stirring for 1 - 2 h, freeze - drying, and pulverizing and sieving.

[0026] Preferably, in the citric acid / EDTA mixed solution, the concentration of citric acid is 0.1 - 0.5 mol / L, and the concentration of EDTA is 0.01 - 0.05 mol / L.

[0027] Preferably, the ultrasonic frequency is 30 - 50 kHz.

[0028] The application of the above - mentioned compound microbial agent compounded with a plant - derived bacteriostatic agent compound preparation in the preparation of an agricultural long - acting bacteriostatic agent.

[0029] Application of the above compound microbial agent compounded with a plant-derived bacteriostatic agent in antibacterial in the process of pineapple or ananas cultivation.

[0030] Beneficial effects The present invention uses Trichoderma harzianum T-22, Bacillus subtilis BS-5 and Pseudomonas fluorescens PF-3 for compounding. Trichoderma harzianum T-22 secretes chitinase to decompose cell walls, Bacillus subtilis BS-5 produces antibacterial lipopeptides to directly inhibit the growth of pathogenic bacteria, and Pseudomonas fluorescens PF-3 synthesizes siderophores to competitively deprive pathogenic bacteria of iron ions. The triple action blocks the infection cycle of pathogenic bacteria. Then, it is compounded with a nano-montmorillonite complex grafted with carboxyl-terminated polyamidoamine, which can significantly enhance the colonization ability of the microbial population in the fermentation product.

[0031] The present invention uses aloe vera leaves and sophora flavescens roots for compounding. The plant-derived bioactive components comprehensively achieve efficient and green control of pineapple wilt disease in the field by targeting and inhibiting the infection of pathogenic bacteria, activating plant immunity, and cooperating with the compound microbial agent for improving the rhizosphere microecology. It shows a synergistic effect within a certain mass ratio range, can improve the control effect on pineapple wilt disease, is beneficial to reducing the dosage of pesticides, and reducing the control cost and environmental pollution. Further, the microencapsulation technology of sodium alginate-chitosan is adopted, so that the slow-release period of the plant active components in the rhizosphere is greatly extended. After the nano-montmorillonite is modified by dendritic carboxyl-terminated polyamidoamine, the specific surface area is greatly increased, significantly enhancing the colonization density of the functional microbial population in the rhizosphere, and the survival rate of the microbial population is significantly improved compared with the conventional carrier.

[0032] The present invention breaks through the bottleneck of low efficiency and short persistence of single biological control through the regulation of functional microbial populations - plant active substances, can delay the generation and development of drug resistance to pineapple wilt disease, can delay the service life of pesticides to a certain extent, and simultaneously improves the soil health index, and is applicable to large-scale field applications. Description of the drawings

[0033] Figure 1 It is a comparison chart of the co-toxicity coefficients of the 5th group of examples, the 1st group of comparative examples, the 2nd group of comparative examples, and the 3rd group of comparative examples.

[0034] Figure 2 It is a comparison chart of the growth rate of urease and phosphatase activities in the soil of the 5th group of examples, the 1st group of comparative examples, the 2nd group of comparative examples, and the 3rd group of comparative examples.

[0035] Figure 3 It is a comparison chart of the root surface density of Pseudomonas fluorescens PF-3 of the 5th group of examples, the 1st group of comparative examples, the 2nd group of comparative examples, and the 3rd group of comparative examples. Detailed implementation manners

[0036] The present invention will be further explained below in combination with specific examples.

[0037] The activated nano-montmorillonite used below is obtained by activating nano-montmorillonite at 125 °C for 90 min.

[0038] Example 1 A composite preparation of a compound bacterial agent and a plant-derived bacteriostatic agent, the raw materials of which include: 70 g of plant-derived bacteriostatic agent, 50 g of fermentation product, 10 g of terminal carboxyl polyamide amine, 40 g of activated nano-montmorillonite, 1 g of azobisisobutyramidine hydrochloride, 10 g of polyvinyl alcohol, and 1 g of silane coupling agent KH-550.

[0039] The plant-derived bacteriostatic agent is prepared by the following steps: adding 10 g of chitosan to 200 g of acetic acid-sodium acetate buffer solution with pH = 5 - 5.5, mixing evenly, adding 10 g of sodium alginate and stirring evenly, adding 50 g of plant-derived bioactive ingredients and stirring evenly, dropping into 1% calcium chloride aqueous solution by peristaltic pump, curing at 5 °C for 10 min, and drying in vacuum.

[0040] Among them, the plant-derived bioactive ingredients used are prepared by the following specific steps: drying and pulverizing aloe vera leaves and sophora flavescens roots respectively, mixing the dried and pulverized aloe vera leaves and the dried and pulverized sophora flavescens roots in a mass ratio of 10:1, drying and pulverizing, sending them into an extraction tank, adding purified water to the extraction tank according to the mass ratio of material to liquid of 1:5, refluxing and extracting at 95 °C for 5 h, and filtering to obtain medicinal residues a and extract a ; concentrating the extract a to a thick paste with a relative density of 1.1 a ; pulverizing the medicinal residues a through a 100-mesh sieve, sending them into an extraction tank, adding 90% ethanol aqueous solution to the extraction tank according to the mass ratio of material to liquid of 1:4, heating and refluxing at 75 °C for 5 h, and filtering to obtain an extract b ; concentrating the extract b under reduced pressure to obtain a thick paste with a relative density of 1.4 b ; mixing the thick pastes a and the thick paste b evenly.

[0041] The fermentation product is prepared by the following specific steps: mixing Trichoderma harzianum T-22, Bacillus subtilis BS-5, and Pseudomonas fluorescens PF-3 evenly to obtain a compound bacterial agent (wherein, the viable count of Trichoderma harzianum T-22 is 2×10 8 CFU / g, the viable count of Bacillus subtilis BS-5 is 1×10 7 CFU / g, and the viable count of Pseudomonas fluorescens PF-3 is 2×10 7 CFU / g); fermenting with bagasse-soybean meal substrate (C / N = 25:1) at 25 °C for 70 h, and the inoculation amount is 3%.

[0042] The preparation method of the above compound microbial agent compounded with a plant-derived bacteriostatic agent includes the following steps: Add terminal carboxyl polyamidoamine to 20 g of a citric acid / EDTA mixed solution and stir evenly. Add activated nano-montmorillonite and perform ultrasonic treatment for 10 min at an ultrasonic frequency of 30 kHz. Adjust the pH value of the system to 7.2 - 7.6, add azobisisobutyramidine hydrochloride, continue ultrasonic treatment for 10 min at a temperature of 50 °C, cool to 30 °C, add the fermentation product, plant-derived bacteriostatic agent, polyvinyl alcohol, and silane coupling agent KH-550, and stir for 1 - 2 h. Then perform freeze-drying and pulverize through a 100-mesh sieve.

[0043] Example 2 A compound microbial agent compounded with a plant-derived bacteriostatic agent, the raw materials of which include: 220 g of plant-derived bacteriostatic agent, 100 g of fermentation product, 30 g of terminal carboxyl polyamidoamine, 60 g of activated nano-montmorillonite, 1 g of azobisisobutyramidine hydrochloride, 30 g of polyvinyl alcohol, and 10 g of silane coupling agent KH-550.

[0044] The plant-derived bacteriostatic agent is prepared by the following steps: Add 10 g of chitosan to 300 g of an acetic acid-sodium acetate buffer solution with a pH of 5 - 5.5 and mix evenly. Add 50 g of sodium alginate and stir evenly. Add 150 g of plant-derived bioactive ingredients and stir evenly. Then, drip the mixture into a 2% calcium chloride aqueous solution through a peristaltic pump and solidify at a temperature of 10 °C for 30 min, followed by vacuum drying.

[0045] Among them, the plant-derived bioactive ingredients are prepared by the following specific steps: Dry and pulverize aloe leaves and sophora flavescens roots respectively. Mix the dried and pulverized aloe leaves and dried and pulverized sophora flavescens roots in a mass ratio of 10:4, dry and pulverize again, and send them into an extraction tank. According to the mass ratio of solid to liquid of 1:10, add purified water to the extraction tank and reflux and extract at a temperature of 100 °C for 10 h, then filter to obtain the medicinal residues a and the extract a , Concentrate the extract a to a thick paste with a relative density of 1.3 a ; Pulverize the medicinal residues a through a 100-mesh sieve, send them into the extraction tank, and according to the mass ratio of solid to liquid of 1:6, add a 95% ethanol aqueous solution to the extraction tank and heat and reflux and extract at a temperature of 80 °C for 15 h, then filter to obtain the extract b , Concentrate the extract b under reduced pressure to obtain a thick paste with a relative density of 1.6 b ; Mix the thick pastes a and the thick paste b evenly.

[0046] The fermentation product is prepared by the following specific steps: Mix Trichoderma harzianum T-22, Bacillus subtilis BS-5, and Pseudomonas fluorescens PF-3 evenly to obtain a compound microbial agent (wherein, the viable count of Trichoderma harzianum T-22 is 2×10 8 CFU / g, the viable count of Bacillus subtilis BS-5 is 5×10 7 CFU / g, and the viable count of Pseudomonas fluorescens PF-3 is 2×10 7 CFU / g); Use bagasse-soybean meal substrate (C / N = 25:1) to ferment at 30°C for 80 h, and the inoculation amount is 3%.

[0047] The preparation method of the above compound microbial agent compounded with a plant-derived bacteriostatic agent composite preparation includes the following steps: Add terminal carboxyl polyamidoamine to 30 g of a citric acid / EDTA mixed solution and stir evenly, add activated nano-montmorillonite and perform ultrasonic treatment for 30 min, and the ultrasonic frequency is 50 kHz; Adjust the pH value of the system to 7.2 - 7.6, add azobisisobutyramidine hydrochloride, continue ultrasonic treatment at 60°C for 30 min, cool to 40°C, add the fermentation product, plant-derived bacteriostatic agent, polyvinyl alcohol, and silane coupling agent KH-550 thereto and stir for 2 h, perform freeze-drying, and pulverize through a 100-mesh sieve.

[0048] Example 3 A compound microbial agent compounded with a plant-derived bacteriostatic agent composite preparation, the raw materials of which include: 120 g of plant-derived bacteriostatic agent, 90 g of fermentation product, 15 g of terminal carboxyl polyamidoamine, 55 g of activated nano-montmorillonite, 1 g of azobisisobutyramidine hydrochloride, 15 g of polyvinyl alcohol, and 7 g of silane coupling agent KH-550.

[0049] The plant-derived bacteriostatic agent is prepared by the following steps: Add 10 g of chitosan to 280 g of an acetic acid-sodium acetate buffer solution with pH = 5 - 5.5 and mix evenly, add 20 g of sodium alginate and stir evenly, add 120 g of plant-derived bioactive ingredients and stir evenly, and drip it into a calcium chloride aqueous solution with a mass fraction of 1.5% through a peristaltic pump, and solidify at 7°C for 25 min, and perform vacuum drying.

[0050] Among them, the plant-derived bioactive ingredients used are prepared by the following specific steps: Dry and pulverize aloe leaves and sophora flavescens roots respectively, mix the dried and pulverized aloe leaves and dried and pulverized sophora flavescens roots in a mass ratio of 10:2, dry and pulverize them, send them into an extraction tank, add purified water to the extraction tank according to a material-liquid mass ratio of 1:9, reflux and extract at 98°C for 7 h, and filter to obtain medicinal residues a and extract a The extract a is concentrated to a thick paste with a relative density of 1.2 a ; The medicinal residues aGround through a 100-mesh sieve and fed into an extraction tank. According to the mass ratio of material to liquid of 1:5.5, an ethanol aqueous solution with a mass fraction of 91% was added to the extraction tank, and heated under reflux at 79 °C for 8 h, and then filtered to obtain an extract. b , and the extract b was concentrated under reduced pressure to obtain a thick paste with a relative density of 1.5. b ; The thick paste a and the thick paste b were mixed evenly.

[0051] The fermentation product was prepared by the following specific steps: Trichoderma harzianum T-22, Bacillus subtilis BS-5, and Pseudomonas fluorescens PF-3 were mixed evenly to obtain a compound microbial agent (wherein, the viable count of Trichoderma harzianum T-22 was 2×10 8 CFU / g, the viable count of Bacillus subtilis BS-5 was 4×10 7 CFU / g, and the viable count of Pseudomonas fluorescens PF-3 was 2×10 7 CFU / g); Fermented with bagasse-soybean meal substrate (C / N = 25:1) at 27 °C for 77 h, and the inoculation amount was 3%.

[0052] The preparation method of the above compound microbial agent compounded with a plant-derived bacteriostatic agent compound preparation includes the following steps: Add terminal carboxyl polyamidoamine to 22 g of a citric acid / EDTA mixed solution and stir evenly, add activated nano-montmorillonite and ultrasonically treat for 25 min, and the ultrasonic frequency is 35 kHz; Adjust the pH value of the system to 7.2 - 7.6, add azobisisobutyramidine hydrochloride, continue to ultrasonically treat at 58 °C for 15 min, cool to 37 °C, add the fermentation product, plant-derived bacteriostatic agent, polyvinyl alcohol, and silane coupling agent KH-550 and stir for 80 min, freeze-dry, and grind through a 100-mesh sieve.

[0053] Example 4 A compound microbial agent compounded with a plant-derived bacteriostatic agent compound preparation, the raw materials thereof include: 190 g of plant-derived bacteriostatic agent, 70 g of fermentation product, 25 g of terminal carboxyl polyamidoamine, 45 g of activated nano-montmorillonite, 1 g of azobisisobutyramidine hydrochloride, 25 g of polyvinyl alcohol, and 3 g of silane coupling agent KH-550.

[0054] The plant-derived bacteriostatic agent was prepared by the following steps: Add 10 g of chitosan to 220 g of an acetic acid-sodium acetate buffer solution with a pH of 5 - 5.5 and mix evenly, add 40 g of sodium alginate and stir evenly, add 80 g of plant-derived bioactive ingredients and stir evenly, and drip into a calcium chloride aqueous solution with a mass fraction of 1.5% through a peristaltic pump, and solidify at 9 °C for 15 min, and then vacuum dry.

[0055] Among them, the plant-derived bioactive ingredients used are prepared by the following specific steps: Aloe vera leaves and Sophora flavescens roots are dried and then crushed. The dried and crushed Aloe vera leaves and the dried and crushed Sophora flavescens roots are mixed in a mass ratio of 10:3, dried and then crushed, and sent into an extraction tank. According to the mass ratio of the material to the liquid of 1:7, purified water is added to the extraction tank, and reflux extraction is carried out at a temperature of 98°C for 9 hours, and the medicinal residues are obtained by filtration. a and the extract a , the extract a is concentrated to a thick paste with a relative density of 1.2 a ; the medicinal residues a are crushed and passed through a 100-mesh sieve, sent into an extraction tank, and according to the mass ratio of the material to the liquid of 1:4.5, an ethanol aqueous solution with a mass fraction of 93% is added to the extraction tank, and heating reflux extraction is carried out at a temperature of 77°C for 12 hours, and the extract is obtained by filtration. b , the extract b is concentrated under reduced pressure to obtain a thick paste with a relative density of 1.5 b ; the thick paste a and the thick paste b are mixed evenly.

[0056] The fermentation product is prepared by the following specific steps: Trichoderma harzianum T-22, Bacillus subtilis BS-5, and Pseudomonas fluorescens PF-3 are mixed evenly to obtain a compound microbial agent (wherein, the viable count of Trichoderma harzianum T-22 is 2×10 8 CFU / g, the viable count of Bacillus subtilis BS-5 is 2×10 7 CFU / g, and the viable count of Pseudomonas fluorescens PF-3 is 2×10 7 CFU / g); Fermentation is carried out for 73 hours at a temperature of 29°C using a bagasse-soybean meal substrate (C / N = 25:1), and the inoculation amount is 3%.

[0057] The preparation method of the above compound microbial agent compounded with a plant-derived bacteriostatic agent composite preparation includes the following steps: Adding carboxyl-terminated polyamidoamine to 28 g of a citric acid / EDTA mixed solution and stirring evenly, adding activated nano-montmorillonite and performing ultrasonic treatment for 15 minutes, with an ultrasonic frequency of 45 kHz; Adjusting the pH value of the system to 7.2 - 7.6, adding azobisisobutyramidine hydrochloride, and continuing ultrasonic treatment at a temperature of 52°C for 25 minutes, cooling to 33°C, adding the fermentation product, the plant-derived bacteriostatic agent, polyvinyl alcohol, and silane coupling agent KH-550 thereto and stirring for 100 minutes, followed by freeze-drying and crushing through a 100-mesh sieve.

[0058] Example 5 A compound microbial agent compounded with a plant-derived bacteriostatic agent composite preparation, the raw materials thereof include: 145 g of a plant-derived bacteriostatic agent, 80 g of a fermentation product, 20 g of carboxyl-terminated polyamidoamine, 50 g of activated nano-montmorillonite, 1 g of azobisisobutyramidine hydrochloride, 20 g of polyvinyl alcohol, and 5 g of silane coupling agent KH-550.

[0059] The plant-derived bacteriostatic agent is prepared by the following steps: Add 10 g of chitosan to 250 g of acetic acid-sodium acetate buffer solution with a pH of 5 - 5.5, mix evenly, add 30 g of sodium alginate and stir evenly, add 100 g of plant-derived bioactive components and stir evenly, then drip it into an aqueous calcium chloride solution with a mass fraction of 1.5% through a peristaltic pump, cure at 8 °C for 20 min, and dry in vacuum.

[0060] Among them, the plant-derived bioactive components are prepared by the following specific steps: Dry and crush aloe vera leaves and sophora flavescens roots respectively, mix the dried and crushed aloe vera leaves and dried and crushed sophora flavescens roots in a mass ratio of 10:2.5, dry and then crush, send them into an extraction tank, add purified water to the extraction tank according to a material-liquid mass ratio of 1:8, reflux and extract at 98 °C for 8 h, and filter to obtain the medicinal residues a and the extract a ; Concentrate the extract a to a thick paste with a relative density of 1.2 a ; Crush the medicinal residues a through a 100-mesh sieve, send them into an extraction tank, add an aqueous ethanol solution with a mass fraction of 92% to the extraction tank according to a material-liquid mass ratio of 1:5, heat and reflux and extract at 78 °C for 10 h, and filter to obtain the extract b ; Concentrate the extract b under reduced pressure to obtain a thick paste with a relative density of 1.5 b ; Mix the thick pastes a and the thick paste b evenly.

[0061] The fermentation product is prepared by the following specific steps: Mix Trichoderma harzianum T-22, Bacillus subtilis BS-5, and Pseudomonas fluorescens PF-3 evenly to obtain a compound microbial agent (where the viable count of Trichoderma harzianum T-22 is 2×10 8 CFU / g, the viable count of Bacillus subtilis BS-5 is 3×10 7 CFU / g, and the viable count of Pseudomonas fluorescens PF-3 is 2×10 7 CFU / g); Ferment with a bagasse-soybean meal substrate (C / N = 25:1) at 28 °C for 75 h, and the inoculation amount is 3%.

[0062] The preparation method of the above-mentioned compound microbial agent compounded with a plant-derived bacteriostatic agent composite preparation comprises the following steps: Add carboxyl-terminated polyamidoamine to 25 g of a citric acid / EDTA mixed solution, stir evenly, add activated nano-montmorillonite, and perform ultrasonic treatment for 20 min at an ultrasonic frequency of 42 kHz; adjust the pH value of the system to 7.2 - 7.6, add azodiisobutyramidine hydrochloride, continue ultrasonic treatment for 20 min at a temperature of 55 °C, cool to 35 °C, add the fermentation product, plant-derived bacteriostatic agent, polyvinyl alcohol, and silane coupling agent KH-550 thereto, stir for 90 min, perform freeze-drying, and pulverize through a 100-mesh sieve.

[0063] Comparative Example 1 A compound microbial agent compounded with a plant-derived bacteriostatic agent composite preparation, the raw materials thereof comprising: 145 g of plant-derived bacteriostatic agent, 80 g of fermentation product, 20 g of carboxyl-terminated polyamidoamine, 50 g of activated nano-montmorillonite, 1 g of azodiisobutyramidine hydrochloride, 20 g of polyvinyl alcohol, and 5 g of silane coupling agent KH-550.

[0064] The plant-derived bacteriostatic agent is prepared by the following steps: Add 10 g of chitosan to 250 g of an acetic acid-sodium acetate buffer solution with a pH of 5 - 5.5, mix evenly, add 30 g of sodium alginate and stir evenly, add 100 g of plant-derived bioactive components and stir evenly, drop the mixture into an aqueous calcium chloride solution with a mass fraction of 1.5% through a peristaltic pump, solidify at a temperature of 8 °C for 20 min, and perform vacuum drying.

[0065] Among them, the plant-derived bioactive components used are prepared by the following specific steps: Dry and pulverize aloe leaves and sophora flavescens roots respectively, mix the dried and pulverized aloe leaves and dried and pulverized sophora flavescens roots in a mass ratio of 10:2.5, dry and pulverize them, send them into an extraction tank, add an ethanol aqueous solution with a mass fraction of 92% to the extraction tank according to a solid-liquid mass ratio of 1:8, reflux and extract at a temperature of 78 °C for 8 h, and filter to obtain the medicinal residues a and the extract a ; concentrate the extract a to a thick paste with a relative density of 1.2 a ; pulverize the medicinal residues a through a 100-mesh sieve, send them into an extraction tank, add an ethanol aqueous solution with a mass fraction of 92% to the extraction tank according to a solid-liquid mass ratio of 1:5, heat and reflux and extract at a temperature of 78 °C for 10 h, and filter to obtain the extract b ; concentrate the extract b under reduced pressure to obtain a thick paste with a relative density of 1.5 b ; mix the thick pastes a and the thick paste b evenly.

[0066] The fermentation product is prepared by the following specific steps: Trichoderma harzianum T-22, Bacillus subtilis BS-5, and Pseudomonas fluorescens PF-3 are mixed evenly to obtain a compound microbial agent (wherein, the viable count of Trichoderma harzianum T-22 is 2×10 8 CFU / g, the viable count of Bacillus subtilis BS-5 is 3×10 7 CFU / g, and the viable count of Pseudomonas fluorescens PF-3 is 2×10 7 CFU / g); bagasse-soybean meal substrate (C / N = 25:1) is fermented at 28°C for 75 h with an inoculation amount of 3%.

[0067] The preparation method of the compound microbial agent compounded with a plant-derived bacteriostatic agent compound preparation includes the following steps: Terminal carboxyl polyamidoamine is added to 25 g of a citric acid / EDTA mixed solution and stirred evenly, activated nano-montmorillonite is added and ultrasonic treated for 20 min with an ultrasonic frequency of 42 kHz; the pH value of the system is adjusted to 7.2 - 7.6, azobisisobutyramidine hydrochloride is added, and ultrasonic treatment is continued at 55°C for 20 min, cooled to 35°C, the fermentation product, plant-derived bacteriostatic agent, polyvinyl alcohol, and silane coupling agent KH-550 are added thereto and stirred for 90 min, freeze-dried, and pulverized through a 100-mesh sieve.

[0068] Comparative Example 2 A compound microbial agent compounded with a plant-derived bacteriostatic agent compound preparation, the raw materials of which include: 100 g of plant-derived bioactive components, 10 g of chitosan, 30 g of sodium alginate, 80 g of fermentation product, 20 g of terminal carboxyl polyamidoamine, 50 g of activated nano-montmorillonite, 1 g of azobisisobutyramidine hydrochloride, 20 g of polyvinyl alcohol, and 5 g of silane coupling agent KH-550.

[0069] Among them, the plant-derived bioactive components used are prepared by the following specific steps: Aloe vera leaves and Sophora flavescens roots are dried and pulverized respectively, the dried and pulverized Aloe vera leaves and dried and pulverized Sophora flavescens roots are mixed according to a mass ratio of 10:2.5, dried and pulverized, sent into an extraction tank, and according to a solid-liquid mass ratio of 1:8, purified water is added to the extraction tank, and reflux extraction is carried out at 98°C for 8 h, and the medicinal residues a and the extract a are obtained. The extract a is concentrated to a thick paste with a relative density of 1.2 a ; the medicinal residues a are pulverized through a 100-mesh sieve, sent into an extraction tank, and according to a solid-liquid mass ratio of 1:5, an ethanol aqueous solution with a mass fraction of 92% is added to the extraction tank, and heating reflux extraction is carried out at 78°C for 10 h, and the extract b is obtained. The extract b is concentrated under reduced pressure to obtain a thick paste with a relative density of 1.5 b ; the thick paste a and the thick pasteb Mix evenly.

[0070] The fermentation product is prepared by the following specific steps: Mix Trichoderma harzianum T-22, Bacillus subtilis BS-5, and Pseudomonas fluorescens PF-3 evenly to obtain a compound microbial agent (wherein, the viable count of Trichoderma harzianum T-22 is 2×10 8 CFU / g, the viable count of Bacillus subtilis BS-5 is 3×10 7 CFU / g, and the viable count of Pseudomonas fluorescens PF-3 is 2×10 7 CFU / g); Use bagasse-soybean meal substrate (C / N = 25:1) to ferment at 28°C for 75 h, and the inoculation amount is 3%.

[0071] The preparation method of the above compound microbial agent compounded with a plant-derived bacteriostatic agent compound preparation includes the following steps: Add terminal carboxyl polyamidoamine to 25 g of citric acid / EDTA mixed solution and stir evenly, add activated nano-montmorillonite and perform ultrasonic treatment for 20 min, and the ultrasonic frequency is 42 kHz; Adjust the pH value of the system to 7.2 - 7.6, add azobisisobutyramidine hydrochloride, continue ultrasonic treatment at 55°C for 20 min, cool to 35°C, add the fermentation product, plant-derived bioactive ingredient, chitosan, sodium alginate, polyvinyl alcohol, and silane coupling agent KH-550 and stir for 90 min, drop it into a 1.5% calcium chloride aqueous solution through a peristaltic pump, solidify at 8°C for 20 min, freeze-dry, and pulverize through a 100-mesh sieve.

[0072] Comparative Example 3 A compound microbial agent compounded with a plant-derived bacteriostatic agent compound preparation, the raw materials of which include: 145 g of plant-derived bacteriostatic agent, 80 g of fermentation product, 20 g of terminal carboxyl polyamidoamine, 50 g of activated nano-montmorillonite, 1 g of azobisisobutyramidine hydrochloride, 20 g of polyvinyl alcohol, and 5 g of silane coupling agent KH-550.

[0073] The plant-derived bacteriostatic agent is prepared by the following steps: Add 10 g of chitosan to 250 g of acetic acid-sodium acetate buffer solution with pH = 5 - 5.5 and mix evenly, add 30 g of sodium alginate and stir evenly, add 100 g of plant-derived bioactive ingredient and stir evenly, drop it into a 1.5% calcium chloride aqueous solution through a peristaltic pump, solidify at 8°C for 20 min, and vacuum dry.

[0074] Among them, the plant-derived bioactive ingredient used is prepared by the following specific steps: Dry and pulverize aloe vera leaves and sophora flavescens roots respectively, mix the dried and pulverized aloe vera leaves and dried and pulverized sophora flavescens roots according to a mass ratio of 10:2.5, dry and pulverize, send them into an extraction tank, add purified water to the extraction tank according to a material-liquid mass ratio of 1:8, reflux and extract at 98°C for 8 h, and filter to obtain the medicinal residues a and the extracta , concentrate the extract a to a thick paste with a relative density of 1.2 a ; pulverize the medicinal residues a through a 100-mesh sieve, put them into an extraction tank, add an ethanol aqueous solution with a mass fraction of 92% to the extraction tank according to the mass ratio of the material liquid of 1:5, heat and reflux for extraction at 78 °C for 10 h, and filter to obtain the extract b , concentrate the extract b under reduced pressure to obtain a thick paste with a relative density of 1.5 b ; mix the thick paste a and the thick paste b evenly.

[0075] The fermentation product is prepared by the following specific steps: uniformly mix Trichoderma harzianum T-22 and Pseudomonas fluorescens PF-3 to obtain a compound microbial agent (wherein, the viable count of Trichoderma harzianum T-22 is 2×10 8 CFU / g, and the viable count of Pseudomonas fluorescens PF-3 is 2×10 7 CFU / g); ferment with a bagasse-soybean meal substrate (C / N = 25:1) at 28 °C for 75 h, and the inoculation amount is 3%.

[0076] The preparation method of the above compound microbial agent compounded with a plant-derived bacteriostatic agent compound preparation includes the following steps: add terminal carboxyl polyamidoamine to 25 g of a citric acid / EDTA mixed solution and stir evenly, add activated nano-montmorillonite and ultrasonically treat for 20 min, and the ultrasonic frequency is 42 kHz; adjust the pH value of the system to 7.2 - 7.6, add azobisisobutyramidine hydrochloride, continue to ultrasonically treat at 55 °C for 20 min, cool to 35 °C, add the fermentation product, the plant-derived bacteriostatic agent, polyvinyl alcohol, and silane coupling agent KH-550 and stir for 90 min, freeze-dry, and pulverize through a 100-mesh sieve.

[0077] Cut fresh leaves of Tainong No. 16 pineapple infected with pineapple wilt virus, add 0.01 mol / L PBS buffer solution with pH = 7.2 (the ratio of the mass of fresh leaves to the volume of PBS buffer solution is 50 mg:1 L), add a little quartz sand with 500 meshes, grind and homogenize, and filter the residue through 4 layers of gauze to obtain the virus solution for standby.

[0078] Select vigorous, healthy Tainong No. 16 pineapple seedlings with 6 leaves in Wenchang, Hainan. Spray carborundum on the surface of each leaf, and gently rub with a brush dipped in the virus solution for rubbing inoculation. After inoculation, gently rinse the surface of the leaf with water and dry it in the air.

[0079] Randomly divided into 7 groups (5 groups of examples, 1 group of comparative example 1, 1 group of comparative example 2, 1 group of comparative example 3, plant-derived bacteriostatic agent group, fermentation product group, blank group), with 3 replicates in each group and 20 seedlings in each replicate. Seven days before transplanting, the compound preparations obtained in Example 5, the compound preparations obtained in Comparative Examples 1-3, the plant-derived bacteriostatic agent obtained in Example 5, the fermentation product obtained in Example 5, and clear water (2 kg / mu) were mixed with organic fertilizer (200 kg / mu) and applied by ditch application; after transplanting, the compound preparations obtained in Example 5, the compound preparations obtained in Comparative Examples 1-3, the plant-derived bacteriostatic agent obtained in Example 5, and the fermentation product obtained in Example 5 (diluted 500 times) were diluted with clear water and then sprayed on the leaves of pineapple seedlings with the aforementioned diluted solution and clear water, once every 5 days for a total of 10 times. The disease incidence was investigated, and the disease index and relative control efficacy were calculated.

[0080]

[0081]

[0082] The disease grade table is shown in Table 1.

[0083]

[0084] Regression analysis was performed on the logarithm of the drug concentration of each treatment and the probability value of the relative control efficacy of each treatment to calculate the EC of the drug for each treatment 50 , and the co-toxicity coefficient (CTC value) of the mixture was calculated according to the Sun Yunpei method.

[0085] According to the calculated co-toxicity coefficient (CTC), the synergistic effect of the drug was evaluated. CTC ≤ 80 was antagonistic effect, 80 < CTC < 120 was additive effect, and CTC ≥ 120 was synergistic effect.

[0086] As Figure 1 shown, the CTCs of the groups of Example 5, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were all greater than 120, indicating that they all formed synergistic effects; while the co-toxicity coefficient of the group of Example 5 was the highest, significantly higher than that of Comparative Examples 1-3 (P < 0.05), confirming that the compound preparation obtained in the present invention had the best control effect on pineapple wilt disease.

[0087] Soils of the groups of Example 5, Comparative Example 1, Comparative Example 2, Comparative Example 3, plant-derived bacteriostatic agent group, fermentation product group, and blank group were sampled, and the urease activity and phosphatase activity of the soils of each group were measured. Based on the blank group, the growth rates of urease and phosphatase activities were calculated.

[0088] As Figure 2 shown, the growth rates of urease and phosphatase activities in the soil of the group of Example 5 were the highest, superior to those of Comparative Examples 1-3 (P < 0.05).

[0089] Clean the soil from the roots of the pineapple seedlings in the Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group, and then immerse them in sterile water for 1 h, and calculate the density of Pseudomonas fluorescens PF-3 on the root surface. As Figure 3 shown, the density of Pseudomonas fluorescens PF-3 on the root surface in the Example 5 group was the highest, which was better than that in the Comparative Example 1-3 groups (P < 0.05).

[0090] The above is only a preferred specific embodiment 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 composite preparation of a composite bacterial agent and a composite botanical antibacterial agent, characterized in that: The raw materials include, by mass: 70-220 parts of plant-derived antibacterial agent, 50-100 parts of fermentation product, 10-30 parts of carboxyl-terminated polyamide amine, 40-60 parts of activated nano-montmorillonite, 1 part of azo initiator, 10-30 parts of binder, and 1-10 parts of silane coupling agent; The plant-derived antibacterial agent is prepared by the following steps: adding chitosan to acetic acid-sodium acetate buffer and mixing evenly, adding sodium alginate and stirring evenly, adding plant-derived bioactive ingredients and stirring evenly, dropping into calcium chloride solution, solidifying, and vacuum drying; The fermentation product is obtained by fermenting bagasse-soybean meal matrix with a composite bacterial agent; the composite bacterial agent includes: Trichoderma harzianum T-22, Bacillus subtilis BS-5, and Pseudomonas fluorescens PF-3; the carbon-nitrogen ratio in the bagasse-soybean meal matrix is ​​24-26:

1.

2. The composite preparation of the composite bacterial agent and the composite botanical antibacterial agent according to claim 1, characterized in that: The mass ratio of the plant-derived bioactive component to chitosan and sodium alginate is 5-15:1:1-5.

3. The composite preparation of composite bacterial agent and plant-derived antibacterial agent according to claim 1, characterized in that: The plant-derived bioactive ingredients are prepared by the following specific steps: Aloe vera leaves and Sophora flavescens roots are dried and crushed, then mixed, and water is used for the first reflux extraction to obtain the medicinal residue a and extract a , the extract a Concentrate to obtain thick paste a ; The residue a The extract was crushed and then subjected to a second reflux extraction with an ethanol aqueous solution to obtain an extract. b , the extract b Concentrate to obtain thick paste b ; Make thick paste a Thick paste b Mix well.

4. The composite preparation of composite bacterial agent and plant-derived antibacterial agent according to claim 3, characterized in that: The mass ratio of the dried and crushed aloe leaves to the dried and crushed Sophora flavescens roots is 10:1-4; During the first reflux extraction process, the material-liquid mass ratio is 1:5-10, the reflux extraction temperature is 95-100°C, and the reflux extraction is 5-10h; During the second reflux extraction, the mass fraction of the ethanol aqueous solution is 90-95%, the material-liquid mass ratio is 1:4-6, the reflux extraction temperature is 75-80°C, and the reflux extraction is 5-15h; Thick paste a The relative density is 1.1-1.3, thick paste b The relative density is 1.4-1.

6.

5. The composite preparation of composite bacterial agent and plant-derived antibacterial agent according to claim 1, characterized in that: The mass fraction of calcium chloride solution is 1-2%; the curing temperature is 5-10°C, and the curing time is 10-30min.

6. The composite preparation of composite bacterial agent and plant-derived antibacterial agent according to claim 1, characterized in that: In the composite bacterial agent, the number of viable bacteria of Trichoderma harzianum T-22 is 1-2×10 8 CFU / g, the number of viable bacteria of Bacillus subtilis BS-5 is 1-5×10 7 CFU / g, the number of viable bacteria of Pseudomonas fluorescens PF-3 is 1-3×10 7 CFU / g; The fermentation temperature is 25-30℃ and the fermentation time is 70-80h.

7. The composite preparation of composite bacterial agent and plant-derived antibacterial agent according to claim 1, characterized in that: The activated nano-montmorillonite is obtained by activating the nano-montmorillonite at 120-130°C for 1-2h; The azo initiator is azobisisobutylamidine hydrochloride; The binder is polyvinyl alcohol; The silane coupling agent is silane coupling agent KH-550.

8. A method for preparing a composite preparation of a composite bacterial agent and a plant-derived antibacterial agent as claimed in any one of claims 1 to 7, characterized in that: The method comprises the following steps: adding carboxyl-terminated polyamidoamine to a citric acid / EDTA mixed solution and stirring evenly, adding activated nano-montmorillonite and ultrasonically treating the mixture for 10-30 minutes, adjusting the pH value of the system to 7.2-7.6, adding an azo initiator, continuing the ultrasonic treatment at 50-60°C for 10-30 minutes, reducing the temperature to 30-40°C, adding a fermentation product, a plant-derived antibacterial agent, a binder and a silane coupling agent, stirring the mixture for 1-2 hours, freeze-drying, and crushing and sieving.

9. Use of the composite preparation of the composite bacterial agent and the composite botanical antibacterial agent as claimed in any one of claims 1 to 7 in the preparation of an agricultural long-acting antibacterial agent.

10. Use of the composite microbial agent and plant-derived antibacterial agent composite preparation as claimed in any one of claims 1 to 7 for antibacterial use in the process of pineapple or pineapple planting.

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