Targeted tobacco fusarium root rot bacteriophage pesticide-fertilizer composition and application thereof

Abstract

The invention relates to the technical field of pesticide-fertilizer compositions, in particular to a bacteriophage pesticide-fertilizer composition targeting tobacco fusarium root rot and application of the bacteriophage pesticide-fertilizer composition, and the bacteriophage pesticide-fertilizer composition comprises the following components in parts by weight: 10-30 parts of a bacteriophage component, 60-85 parts of a nutritional component and 5-15 parts of a functional aid, the nutrient components comprise 15-25 parts of a nitrogen source, 10-20 parts of a phosphorus source, 15-30 parts of a potassium source and 5-10 parts of medium trace elements. The bacteriophage pesticide-fertilizer composition for targeting tobacco fusarium root rot is high in targeting property and free of residues and resistance, the bacteriophage can specifically recognize and split target fusarium, other beneficial microorganisms are not damaged, the bacteriophage naturally disappears along with reduction of the number of pathogenic bacteria, no chemical residues exist, the pathogenic bacteria are not prone to being induced to generate resistance, and the pesticide-fertilizer composition is safe and reliable. The pesticide-fertilizer synergistic dual effect is achieved, the fertilizer is retained through pesticide, root diseases are effectively controlled, root health is guaranteed, and therefore the fertilizer utilization rate is remarkably increased.

Description

Technical Field

[0001] This invention relates to the field of pesticide-fertilizer composition technology, specifically to a bacteriophage pesticide-fertilizer composition targeting tobacco sickle root rot and its application. Background Technology

[0002] Tobacco is one of my country's important economic crops. Tobacco falc root rot is a soil-borne fungal disease caused by *Fusarium oxysporum*, and it occurs widely in all tobacco-growing areas of China. This disease primarily infects the tobacco root system, leading to root rot, browning of the vascular bundles, and subsequently wilting and yellowing of the plant. In severe cases, it can cause the entire plant to die, significantly reducing tobacco yield and quality. Statistics show that the incidence rate in severely affected fields can reach 30%-50%, with yield losses exceeding 20%, causing huge economic losses to the tobacco industry.

[0003] Currently, the main methods for controlling tobacco sickle root rot include chemical control, agricultural control, and biological control. Agricultural control measures, such as crop rotation and soil disinfection, are complex and time-consuming, making them difficult to adapt to the needs of large-scale planting. Chemical agents, such as carbendazim and thiophanate-methyl, can suppress the disease in the short term, but long-term use can easily lead to drug resistance in pathogens, and residual chemicals can pollute the soil environment and harm human health, which does not meet the requirements of green agricultural development. Existing biological control methods mostly use biocontrol bacteria or bacteriophages alone, which have problems such as unstable efficacy and short duration of effect, and fail to be synergistically combined with the nutrients required for tobacco growth, making it difficult to achieve the dual goals of disease prevention and growth promotion at the same time.

[0004] Meanwhile, traditional tobacco fertilization mostly uses conventional nitrogen, phosphorus, and potassium fertilizers, which suffer from uneven nutrient release, low utilization rate, and inability to specifically address soil-borne diseases. Although some pesticide-fertilizer combinations attempt to combine bactericides with fertilizers, they suffer from poor compatibility between bactericides and nutrient components, easy decomposition and inactivation of active ingredients, and significant damage to beneficial microorganisms, failing to achieve synergistic effects in disease prevention, growth promotion, and soil improvement.

[0005] Based on this, the present invention provides a phage-based fertilizer composition targeting tobacco sickle root rot and its application, in order to solve the above-mentioned technical problems. Summary of the Invention

[0006] The purpose of this invention is to provide a phage-based fertilizer composition targeting tobacco sickle root rot and its application. By screening specific phage strains and optimizing the ratio of nutrient components and functional adjuvants and the preparation process, the invention achieves a synergistic integration of the disease prevention function of phages and the nutritional function of fertilizers, thus solving the technical problems of unstable efficacy, low nutrient utilization and environmental pollution in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes a phage-based fertilizer composition targeting tobacco sickle root rot, comprising the following components by weight: 10-30 parts of phage component, 60-85 parts of nutrient component, and 5-15 parts of functional adjuvant. The nutritional components include: 15-25 parts nitrogen source, 10-20 parts phosphorus source, 15-30 parts potassium source, and 5-10 parts trace elements. The nitrogen source is composed of a mixture of compound amino acids and coated lysine in a mass ratio of 3:1, and the compound amino acids are composed of glutamic acid and aspartic acid in a mass ratio of 2:1. The phosphorus source is potassium dihydrogen phosphate; the potassium source is potassium sulfate. The trace elements are prepared by chelation process of calcium chloride, magnesium sulfate, zinc sulfate and boric acid in a mass ratio of 4:3:2:1 to obtain EDTA-citric acid composite chelate; The functional adjuvants include 2-5 parts of phage protectant, 3-10 parts of soil conditioner, 5-10 parts of carrier and adsorbent, and 0-3 parts of adhesive. The phage protectant is composed of trehalose, skim milk powder, and glycerol in a mass ratio of 2:1:0.5. The soil conditioner is composed of humic acid, tobacco straw-based biochar, and chitosan in a mass ratio of 3:2:1. The carrier and adsorbent are attapulgite clay activated with hydrochloric acid; The adhesive is either xanthan gum or soluble starch.

[0008] Preferably, the method for preparing the phage component is as follows: A1. Collect soil samples from the 0-20cm rhizosphere layer of tobacco fields affected by Fusarium root rot, with each sample weighing 50g. Add the soil samples to LB enrichment medium containing Fusarium oxysporum inoculum, with an initial concentration of 1×10⁻⁶. 6 The culture medium was prepared with CFU / mL, and 0.5% glucose, 0.3% yeast extract, and 0.1% calcium chloride were added. The medium was then incubated at 30℃ and 180 rpm for 24 h with shaking. After centrifugation at 8000 rpm for 20 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the phage enrichment solution. A2. The phage enrichment solution was separated using the double-layer plate method. The lower layer was LB medium containing 2% agar, and the upper layer was LB medium containing 0.7% agar. The phage enrichment solution was serially diluted and mixed with Fusarium oxysporum bacterial suspension. The mixture was then evenly spread on the upper layer of medium and incubated at 30°C for 12-16 hours. Clear plaques were picked and the purification was repeated 3-5 times to obtain a single phage strain. A3. Through morphological observation, 16S rRNA gene sequencing and fragmentation spectrum determination, two different strains with fragmentation rate ≥90% and strong host specificity were screened out and named FoP1 strain and FoP2 strain, respectively. A4. Using LB medium as a base, a fermentation medium was constructed by adding 0.5% glucose and 0.3% yeast extract. A single phage strain was inoculated and fermented for 18-24 hours at a temperature of 30-32℃, pH 7.0-7.2, aeration rate of 1.0-1.2 vvm, and stirring speed of 200-250 r / min. After fermentation, the phage was concentrated by centrifugation at 8000 r / min for 20 min to obtain a titer of not less than 1×10¹. 0 A single-strain concentrate with PFU / mL was prepared by mixing FoP1 and FoP2 strains at a mass ratio of 1:1-3. The final phage component titer was 5 × 10⁻⁶. 9 -1×10¹ 0 PFU / mL.

[0009] Preferably, the method for preparing coated lysine in the nitrogen source is as follows: using lysine as the core material, sodium alginate and chitosan at a mass ratio of 2:1 as coating materials, a double-layer coating process is adopted, and coating is carried out at 45-50℃ and stirring speed of 60-80r / min for 2-3 hours to obtain coated lysine with a coating rate of ≥85%.

[0010] Preferably, the preparation method of the phosphorus source potassium dihydrogen phosphate is as follows: potassium dihydrogen phosphate is purified by recrystallization. Potassium dihydrogen phosphate is dissolved in deionized water at 80-90℃ to prepare a solution with a mass fraction of 30-40%. After stirring and dissolving, the solution is kept at the temperature and allowed to stand for 4-6 hours. After filtration and cooling for crystallization, the solution is separated by centrifugation and dried at 105℃ for 3 hours to obtain potassium dihydrogen phosphate with a purity ≥99.5% and an impurity content ≤0.1%. The method for preparing potassium sulfate is as follows: potassium sulfate is pulverized to 100-120 mesh, dried at 105℃ for 2 hours to remove moisture, and potassium sulfate with K2O content ≥50% and moisture content ≤0.5% is obtained.

[0011] Preferably, the method for preparing the trace elements is as follows: calcium chloride, magnesium sulfate, zinc sulfate, and boric acid are weighed in a mass ratio of 4:3:2:1, dissolved in deionized water, and the total concentration of metal ions is controlled at 0.2 mol / L. EDTA and citric acid are added, and the molar ratio of EDTA to citric acid is controlled at 1:0.5. The pH is adjusted to 5.5-6.5, and the mixture is chelated at 60-70℃ and a stirring speed of 100-120 r / min for 3-4 hours to obtain an EDTA-citric acid composite chelate.

[0012] Preferably, the phage protectant is prepared by weighing trehalose, skim milk powder, and glycerol in a mass ratio of 2:1:0.5, mixing them at 10-15℃ and a stirring speed of 50-60 r / min for 20-30 min to obtain a homogeneous mixture.

[0013] Preferably, the soil conditioner is prepared by: crushing tobacco stalks to 20-40 mesh and pyrolyzing them under anaerobic conditions at 500-600℃ for 3-4 hours to obtain tobacco stalk-based biochar with a specific surface area ≥300m² / g; weighing humic acid, tobacco stalk-based biochar, and chitosan at a mass ratio of 3:2:1, and mixing them at 25-30℃ and a stirring speed of 80-100r / min for 30 minutes to obtain the soil conditioner.

[0014] Preferably, the carrier is prepared by: taking attapulgite raw material, soaking it in a 5% hydrochloric acid solution, controlling the liquid-solid ratio at 5:1, activating it at 80℃ and stirring speed at 60-80 r / min for 2 hours, washing it with deionized water until pH 6.5-7.5, drying it at 110℃ for 4 hours, and pulverizing it to 80-100 mesh to obtain activated attapulgite with a specific surface area ≥150 m² / g and an adsorption capacity ≥80 mg / g.

[0015] Preferably, the method for preparing the fertilizer-pesticide composition includes the following steps: S1. Preparation of phage-carrier adsorption solution: Take the phage components prepared in steps A1 to A4 and mix them with the phage protectant at a mass ratio of 5:1 to obtain the phage protectant solution; mix the phage protectant solution with activated attapulgite at a mass ratio of 1:2-3, and adsorb for 60-90 min at 15-20℃ and a stirring speed of 80-100 r / min to obtain wet material; S2. Preparation of nutrient substrate: Take 15-25 parts of nitrogen source, 10-20 parts of phosphorus source, 15-30 parts of potassium source, and 5-10 parts of trace elements, and mix them at 30-40℃ and 150-200r / min for 30-40min to obtain the nutrient substrate. S3. Preparation of composite additive: Take 3-10 parts of soil conditioner and 0-3 parts of adhesive, stir at 25-30℃ and 100-120r / min for 20-30min to obtain composite additive; S4. Mixing and granulation: Mix the wet material obtained in step S1, the nutrient base material obtained in step S2, and the composite additive obtained in step S3, add deionized water to make the material moisture content 15-20%, put it into the granulator, adjust the drum speed to 30-40 r / min, the drum inclination angle to 3-5°, and the material residence time to 15-20 min to make granules of 2.8-4.2 mm; S5. Drying and sieving: The particles obtained in step S4 are dried in stages. The first stage is dried at 40°C for 60 minutes, and the second stage is dried at 45°C for 30 minutes. The drying is carried out until the moisture content is ≤5%. After sieving through 2.8 mm and 4.2 mm standard sieves, the phage fertilizer composition is obtained.

[0016] Based on the above composition, the present invention also proposes the application of a phage fertilizer composition targeting tobacco sickle root rot in the prevention and control of tobacco sickle root rot and the promotion of tobacco plant growth.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention presents a phage-based fertilizer-pesticide composition targeting tobacco falcatella root rot. It exhibits strong targeting, leaves no residue, and does not induce resistance. The phage specifically recognizes and lyses the target Fusarium, without harming other beneficial microorganisms. It naturally dies off as the number of pathogens decreases, leaving no chemical residue and unlikely to induce resistance in pathogens. This achieves a synergistic effect of pesticide and fertilizer, effectively controlling root diseases and ensuring root health by protecting fertilizer. This significantly improves fertilizer utilization. The fertilizer promotes pesticide application, providing ample nutrition to promote robust tobacco plant growth and enhance its disease resistance. Combined with the external control provided by the phage, it creates a synergistic effect of both internal and external prevention. The effect is stable and rapid. Under suitable conditions, the phage can self-reproduce. This product rapidly increases the number of pathogens at disease sites, controlling pests faster than ordinary biological agents. It is easy to operate, cost-effective, and efficient: it can be applied directly using existing fertilization machinery, greatly reducing labor intensity and time costs, achieving true integrated application of pesticides and fertilizers with simplified operations. It provides pre-emptive control, creating a protective zone containing bacteriophages and nutrients in the soil before tobacco root development and disease occurrence, enabling earlier biological intervention and nutrient supply, resulting in better disease prevention and growth promotion effects, soil improvement, and ecological benefits. The addition of functional adjuvants helps improve the rhizosphere soil environment, promotes the construction of beneficial microbial communities, and achieves green and sustainable agricultural production. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] I. Materials: Unless otherwise specified, all components of the phage-based fertilizer composition for targeting tobacco sickle root rot in this invention are commercially available. It is composed of the following raw materials in parts by weight: 10-30 parts of phage component, 60-85 parts of nutrient component, and 5-15 parts of functional adjuvant; It should be noted that the nutritional components include: 15-25 parts nitrogen source, 10-20 parts phosphorus source, 15-30 parts potassium source, and 5-10 parts trace elements; The nitrogen source is a mixture of complex amino acids and coated lysine in a mass ratio of 3:1, and the complex amino acids are a mixture of glutamic acid and aspartic acid in a mass ratio of 2:1. The phosphorus source is potassium dihydrogen phosphate; The potassium source is potassium sulfate; The trace elements were prepared by chelation process of calcium chloride, magnesium sulfate, zinc sulfate and boric acid in a mass ratio of 4:3:2:1 to obtain EDTA-citric acid composite chelate; It should be noted that the functional adjuvants include 2-5 parts of phage protectant, 3-10 parts of soil conditioner, 5-10 parts of carrier and adsorbent, and 0-3 parts of adhesive. The bacteriophage protectant is composed of trehalose, skim milk powder, and glycerin in a mass ratio of 2:1:0.5. The soil conditioner is a mixture of humic acid, tobacco straw-based biochar, and chitosan in a mass ratio of 3:2:1. The carrier and adsorbent are attapulgite clay activated with hydrochloric acid; the adhesive is either xanthan gum or soluble starch. It should be noted that the phage component exhibits specific lytic activity against Fusarium oxysporum, with a titer of not less than 1×10⁻⁶. 8 PFU / mL is composed of one or two of the FoP1 and FoP2 strains in a mass ratio of 1:1-3; both FoP1 and FoP2 strains are specific bacteriophage strains isolated and screened from the rhizosphere soil of tobacco fields infected with tobacco falcon root rot, with a lysis rate of ≥90% against Fusarium oxysporum. II. Process: Based on the above formulation, this invention also proposes a method for preparing a phage-based fertilizer composition targeting tobacco sickle root rot, specifically including the following steps: The method for preparing bacteriophage components includes the following steps: A1. Sampling and Enrichment Culture: Soil samples were collected from the rhizosphere layer (0-20 cm) of tobacco fields affected by Fusarium root rot, with each sample weighing 50 g. The soil samples were then added to LB enrichment medium containing Fusarium oxysporum inoculum, with an initial concentration of 1 × 10⁻⁶. 6 CFU / mL, the culture medium was supplemented with 0.5% glucose, 0.3% yeast extract and 0.1% calcium chloride by mass fraction, and cultured with shaking at 30℃ and 180r / min for 24h. Then, it was centrifuged at 8000r / min for 20min, and the supernatant was filtered through a 0.22μm filter membrane to obtain the phage enrichment solution. A2. Isolation and purification: The double-layer plate method was used for isolation. The lower culture medium was LB medium containing 2% agar, and the upper culture medium was LB medium containing 0.7% agar. The phage enrichment solution was serially diluted and mixed with Fusarium oxysporum bacterial suspension. The mixture was evenly spread on the upper culture medium and incubated at 30℃ for 12-16 h. Clear phage plaques were picked and the purification was repeated 3-5 times to obtain a single phage strain. A3. Strain identification: Through morphological observation (transmission electron microscopy), 16S rRNA gene sequencing, and fragmentation spectrum determination, two different strains with a fragmentation rate ≥90% and strong host specificity were screened out and named FoP1 strain and FoP2 strain, respectively. Both FoP1 and FoP2 strains are Gram-negative bacteriophages, with a tadpole-shaped morphology, a head diameter of 50-70 nm, a tail length of 100-150 nm, and a double-stranded DNA genome. They do not lyse beneficial microorganisms such as Bacillus subtilis and Trichoderma in the soil. A4. Fermentation and Concentration: A fermentation medium was constructed using LB medium supplemented with 0.5% glucose and 0.3% yeast extract. A single phage strain was inoculated and fermented for 18-24 hours at 30-32℃, pH 7.0-7.2, aeration rate of 1.0-1.2 vvm, and stirring speed of 200-250 r / min. After fermentation, the phage was concentrated by centrifugation at 8000 r / min for 20 min to obtain a titer of not less than 1×10¹. 0 A single-strain concentrate with PFU / mL; after mixing FoP1 and FoP2 strains at a mass ratio of 1:1-3, the final phage component titer was 5×10. 9 -1×10¹ 0 PFU / mL; Based on this, the phage component exhibits specific lytic activity against Fusarium oxysporum, with a titer of not less than 1×10⁻⁶. 8 PFU / mL is composed of one or two of the FoP1 and FoP2 strains in a mass ratio of 1:1-3; both FoP1 and FoP2 strains are specific bacteriophage strains isolated and screened from the rhizosphere soil of tobacco fields infected with tobacco falcon root rot, with a lysis rate of ≥90% against Fusarium oxysporum. Both FoP1 and FoP2 strains are Gram-negative bacteriophages, tadpole-shaped with a head diameter of 50-70 nm and a tail length of 100-150 nm. Their genomes are double-stranded DNA. They exhibit a lysis rate of ≥90% against both the standard strain of Fusarium oxysporum and tobacco field isolates. Their host range is limited to the genus Fusarium spp., and they do not lyse beneficial microorganisms such as Bacillus subtilis and Trichoderma in the soil. A nitrogen source preparation method includes the following steps: using lysine as the core material, and sodium alginate and chitosan at a mass ratio of 2:1 as coating materials, a double-layer coating process is adopted, and coating is carried out at 45-50℃ and a stirring speed of 60-80 r / min for 2-3 hours to obtain coated lysine with a coating rate ≥85%; glutamic acid and aspartic acid are compounded at a mass ratio of 2:1 to obtain a composite amino acid, and then the composite amino acid is mixed with the coated lysine at a mass ratio of 3:1 to obtain the nitrogen source; wherein the purity of glutamic acid and aspartic acid is ≥98%, and the purity of lysine is ≥99%; The preparation methods for phosphorus and potassium sources include the following steps: Phosphorus source is purified by recrystallization. Potassium dihydrogen phosphate is dissolved in deionized water at 80-90℃ to prepare a solution with a mass fraction of 30-40%. After stirring and dissolving, the solution is kept at a constant temperature for 4-6 hours, filtered, cooled, and crystallized. After centrifugation, the solution is dried at 105℃ for 3 hours to obtain potassium dihydrogen phosphate with a purity ≥99.5% and an impurity content ≤0.1%. Potassium source is prepared by pulverizing potassium sulfate to 100-120 mesh and drying it at 105℃ for 2 hours to remove moisture, resulting in potassium sulfate with a K2O content ≥50% and a moisture content ≤0.5%. A method for preparing trace elements includes the following steps: Calcium chloride, magnesium sulfate, zinc sulfate, and boric acid are weighed in a mass ratio of 4:3:2:1, dissolved in deionized water, and the total metal ion concentration is controlled at 0.2 mol / L. EDTA and citric acid are added, and the molar ratio of EDTA to citric acid is controlled at 1:0.5. The pH is adjusted to 5.5-6.5, and the mixture is chelated at 60-70℃ and a stirring speed of 100-120 r / min for 3-4 hours to obtain an EDTA-citric acid composite chelate. The chelate contains ≥12% calcium chelate, ≥8% magnesium chelate, ≥10% zinc chelate, and ≥5% boron chelate, with a chelation rate ≥90%. A method for preparing a bacteriophage protectant includes the following steps: weighing trehalose, skim milk powder, and glycerol in a mass ratio of 2:1:0.5, mixing them at 10-15℃ and a stirring speed of 50-60 r / min for 20-30 min to obtain a homogeneous mixture; wherein the purity of trehalose is ≥99%, the protein content of skim milk powder is ≥80%, and the purity of glycerol is ≥99.5%; A method for preparing a soil conditioner includes the following steps: tobacco straw is pulverized to 20-40 mesh and pyrolyzed under anaerobic conditions at 500-600℃ for 3-4 hours to obtain tobacco straw-based biochar with a specific surface area ≥300m² / g; humic acid, tobacco straw-based biochar, and chitosan are weighed at a mass ratio of 3:2:1 and mixed at 25-30℃ and a stirring speed of 80-100r / min for 30 minutes to obtain the soil conditioner; wherein the humic acid content is ≥70% and the degree of deacetylation of chitosan is ≥85%; The carrier preparation method includes the following steps: taking attapulgite raw material, soaking it in a 5% hydrochloric acid solution, controlling the liquid-solid ratio at 5:1, activating it at 80℃ and stirring speed at 60-80 r / min for 2 hours, washing it with deionized water until pH 6.5-7.5, drying it at 110℃ for 4 hours, and pulverizing it to 80-100 mesh to obtain activated attapulgite with a specific surface area ≥150 m² / g and an adsorption capacity ≥80 mg / g; The adhesive pretreatment method includes the following steps: when the adhesive is xanthan gum, ensure its purity is ≥98% and the viscosity of its 1% aqueous solution is ≥1500 mPa·s at 25℃; when the adhesive is soluble starch, ensure its purity is ≥99% and the gelatinization temperature is 60-65℃; add 1-3 parts for later use. Based on the above material components, the preparation process of the phage-based fertilizer composition targeting tobacco sickle root rot of the present invention specifically includes the following steps: S1. Preparation of phage-carrier adsorption solution: Take the phage components prepared in steps A1 to A4 and mix them with the phage protectant at a mass ratio of 5:1 to obtain the phage protectant solution; mix the phage protectant solution with activated attapulgite at a mass ratio of 1:2-3, and adsorb for 60-90 min at 15-20℃ and a stirring speed of 80-100 r / min to obtain wet material; S2. Preparation of nutrient substrate: Take 15-25 parts of nitrogen source, 10-20 parts of phosphorus source, 15-30 parts of potassium source, and 5-10 parts of trace elements, and mix them at 30-40℃ and 150-200r / min for 30-40min to obtain the nutrient substrate. S3. Preparation of composite additive: Take 3-10 parts of soil conditioner and 0-3 parts of adhesive, stir at 25-30℃ and 100-120r / min for 20-30min to obtain composite additive; S4. Mixing and granulation: Mix the wet material obtained in step S1, the nutrient base material obtained in step S2, and the composite additive obtained in step S3. Add deionized water to make the material moisture content 15-20%. Put the mixture into a granulator, adjust the drum speed to 30-40 r / min, the drum inclination angle to 3-5°, and the material residence time to 15-20 min to make granules of 2.8-4.2 mm. S5. Drying and sieving: The particles obtained in step S4 are dried in stages. The first stage is dried at 40°C for 60 minutes, and the second stage is dried at 45°C for 30 minutes. The moisture content is dried to ≤5%. After sieving through 2.8 mm and 4.2 mm standard sieves, the phage fertilizer composition targeting tobacco sickle root rot is obtained. The present invention will be further described in detail below with reference to specific embodiments. The raw materials used in the embodiments are all commercially available conventional raw materials, and the process parameters unless otherwise specified are performed according to the above-mentioned general process: Example 1: In this example, a bacteriophage-based fertilizer composition targeting tobacco sickle root rot was prepared according to the following process. The formulation components, by weight, include: Twenty phage fractions were prepared, including a FoP1 strain and a FoP2 strain mixed at a mass ratio of 1:2, with a titer of 8 × 10⁻⁶. 9 PFU / mL; Nutritional components: 72 parts: 20 parts nitrogen source, including 15 parts compound amino acids plus 5 parts coated lysine, the compound amino acids being a mixture of 10 parts glutamic acid and 5 parts aspartic acid; 15 parts phosphorus source; 22 parts potassium source; 5 parts trace elements, including 2 parts calcium chloride plus 1.5 parts magnesium sulfate plus 1 part zinc sulfate plus 0.5 parts boric acid, which are chelated together. Functional additives (8 parts): phage protectant (3 parts, including 2 parts trehalose, 1 part skim milk powder, and 0.5 parts glycerin); soil conditioner (3 parts, including 1.5 parts humic acid, 1 part tobacco straw-based biochar, and 0.5 parts chitosan); carrier and adsorbent (2 parts); and adhesive (0 parts). The preparation method includes the following steps: S1. Preparation of each component: Prepare phage components, nitrogen source, phosphorus source, potassium source, trace elements, phage protectant, soil conditioner, carrier and adsorbent according to the above general process to ensure that each performance indicator meets the standard; S2. Preparation of phage-carrier adsorption solution: Take 20 parts of phage component and mix with 3 parts of phage protectant to obtain phage protectant solution; mix the phage protectant solution with 2 parts of activated attapulgite clay, and adsorb for 75 min at 18℃ and stirring speed of 90 r / min to obtain wet material; S3. Preparation of nutrient substrate: Take 20 parts of nitrogen source, 15 parts of phosphorus source, 22 parts of potassium source, and 5 parts of trace elements, mix them at 35℃ and stirring speed of 180r / min for 35min to obtain nutrient substrate; S4. Preparation of composite additive: Take 3 parts of soil conditioner and 0 parts of adhesive and mix them. Stir at 28℃ and 110r / min for 25min to obtain the composite additive. S5. Mixing and granulation: Mix the wet material, nutrient base material, and compound additives, add deionized water to make the material moisture content 18%, put it into the granulator, adjust the drum speed to 35r / min, the drum inclination angle to 4°, and the material residence time to 18min to make 3.5mm granules; S6. Drying and sieving: The granules are dried in stages. The first stage is dried at 40℃ for 60 minutes, and the second stage is dried at 45℃ for 30 minutes. The moisture content is dried to 4%. After sieving through 2.8 mm and 4.2 mm standard sieves, the phage fertilizer composition is obtained. Example 2, in this example, the formulation components include, by weight: Ten phage fractions, FoP1 strain only, titer 1×10¹ 0 PFU / mL; The nutritional components consist of 85 parts: 15 parts nitrogen source, including 11.25 parts compound amino acids plus 3.75 parts coated lysine, which is a compound amino acid mixture of 7.5 parts glutamic acid and 3.75 parts aspartic acid; 20 parts phosphorus source; 30 parts potassium source; and 10 parts trace elements, including 4 parts calcium chloride, 3 parts magnesium sulfate, 2 parts zinc sulfate, and 1 part boric acid, which are chelated together. Five functional additives: two parts of phage protectant, including 1.33 parts of trehalose, 0.67 parts of skim milk powder, and 0.33 parts of glycerol; three parts of soil conditioner, including 1.5 parts of humic acid, 1 part of tobacco straw-based biochar, and 0.5 parts of chitosan; 0 parts of carrier and adsorbent; and 0 parts of adhesive. Preparation steps: Except for adjusting the amount of each component according to the above ratio, the other process parameters are the same as in Example 1; Example 3: In this example, the formulation components, by weight, include: Thirty phage fractions were collected, in which the FoP1 and FoP2 strains were mixed at a mass ratio of 1:3, with a titer of 1×10¹. 0 PFU / mL; Nutritional components (60 parts): 25 parts nitrogen source, including 18.75 parts complex amino acids plus 6.25 parts coated lysine, the complex amino acids being a compound of 12.5 parts glutamic acid and 6.25 parts aspartic acid; 10 parts phosphorus source; 15 parts potassium source; and 10 parts trace elements, including 4 parts calcium chloride plus 3 parts magnesium sulfate plus 2 parts zinc sulfate plus 1 part boric acid, which are chelated together. 15 parts of functional additives: 5 parts of phage protectant, including 3.33 parts of trehalose, 1.67 parts of skim milk powder, and 0.83 parts of glycerol; 10 parts of soil conditioner, including 5 parts of humic acid, 3.33 parts of tobacco straw-based biochar, and 1.67 parts of chitosan; 0 parts of carrier and adsorbent; and 0 parts of adhesive. Preparation steps: Except for adjusting the amount of each component according to the above ratio, the other process parameters are the same as in Example 1; Comparative Example 1: Compared with Example 1, only the mixing ratio of phage strains was changed. FoP1 strain and FoP2 strain were mixed at a mass ratio of 1:4. All other formulation components and preparation processes were the same as in Example 1. Comparative Example 2, compared with Example 1, removed the phage component, while the remaining formulation components and preparation process were the same as in Example 1, and it was just a conventional fertilizer; Comparative Example 3, compared with Example 1, removed the coated lysine from the nutrient components, and the nitrogen source was only the complex amino acid. The remaining formulation components and preparation process were the same as in Example 1. Comparative Example 4 used commercially available tobacco-specific fertilizer containing the chemical fungicide carbendazim, with an effective ingredient content of 20%. III. Performance Testing: The performance of the pesticide-fertilizer compositions prepared in Examples 1-3 and Comparative Examples 1-4 was tested. The test items included the control effect on tobacco sickle root rot, tobacco growth indicators, and soil indicators. The test standards and methods are as follows: a. Control efficacy test: Referring to the "Guidelines for Field Efficacy Tests of Pesticides (I) Control of Tobacco Root Rot with Fungicides" (GB / T17980.102-2004), field trials were conducted in tobacco fields with a high incidence of tobacco sickle root rot. The plot area was 20m², with 3 replicates and randomized block design. The soil application method was used, with a dosage of 30 kg per mu, applied one week before tobacco transplanting; the disease incidence was investigated 60 days after transplanting, and the control effect was calculated. Prevention and control efficacy (%) = (Incidence rate in the blank control area - Incidence rate in the treatment area) / Incidence rate in the blank control area × 100%; No pesticides or fertilizers were applied to the blank control area; b. Growth index detection: 60 days after transplanting, 10 tobacco plants were randomly selected from each plot to measure plant height (accurate to 0.1 cm) and maximum leaf area (accurate to 0.1 cm²) using a leaf area meter. The above-ground fresh weight was weighed using an electronic balance (accurate to 0.1 g). The average value was calculated. c. Soil index testing: 60 days after application, soil samples were collected from the 0-20cm soil layer of each plot. Soil pH was determined according to "Soil Testing Part 2: Determination of Soil pH" (NY / T1121.2-2006); the number of Bacillus subtilis in the soil was determined according to "Methods for Determination of Soil Microbial Biomass" using the plate count method. d. Safety testing: Observe whether pesticide damage symptoms appear during the tobacco growth process, including yellowing, wilting, and deformity, and assess product safety; Performance data for the examples and comparative examples are shown in Table 1: IV. Analysis Conclusion: As shown in Table 1, the phage fertilizer compositions prepared in Examples 1-3 of this invention exhibit excellent performance in controlling tobacco sickle root rot and promoting tobacco growth, and are also friendly to the soil microenvironment. Please refer to Table 1. The control efficacy of Examples 1-3 is all above 80%, with Example 3 achieving the highest control efficacy of 88.4%. However, the control efficacy of Comparative Example 1 dropped to 73.0% because the mixing ratio of bacteriophage strains exceeded the 1:1-3 range specified in this invention, indicating that a reasonable ratio of bacteriophage strains is key to ensuring control efficacy. The control efficacy of Comparative Example 2 (without bacteriophage components) was only 26.8%, significantly lower than the examples, verifying the specific lytic effect of bacteriophage components on Fusarium oxysporum. The control efficacy of Comparative Example 4 (commercially available chemical fertilizer) was 76.7%, lower than the examples of this invention, and showed slight phytotoxicity, while the examples of this invention showed no phytotoxicity and were safer. Please refer to Table 1. The tobacco plant height, maximum leaf area, and aboveground fresh weight of Examples 1-3 were significantly higher than those of the comparative example and the blank control. Among them, the plant height of Example 3 reached 60.2 cm and the aboveground fresh weight reached 192.4 g / plant. Due to the removal of the coating lysine, the nutrient release of Comparative Example 3 was uneven, and its growth indicators were slightly lower than those of Example 1, indicating that the addition of coating lysine can improve nutrient utilization and promote tobacco growth. The growth indicators of Comparative Example 2 (with only conventional fertilizer) were poor, which verifies the synergistic growth-promoting effect of the nutrient components of this invention. Please refer to Table 1. The soil pH in Examples 1-3 was maintained at 6.7-6.9, which is suitable for tobacco growth, and the number of Bacillus subtilis in the soil was all above 2.5 × 10⁻⁶. 6 The cfu / g soil concentration was significantly higher than that of Comparative Example 4, indicating that the composition of the present invention does not damage the beneficial microbial community in the soil and can improve the soil micro-ecological environment; while Comparative Example 4 resulted in a reduction in the number of beneficial microorganisms in the soil, and long-term use could easily cause soil compaction. In summary, the formulation component ranges of Examples 1-3 of this invention are scientifically reasonable, and the synergistic effect of each component is significant. Among them, the formulation and process parameters of Example 1 are balanced, which has both excellent prevention and control effects and growth promotion effects, and the preparation cost is relatively low, making it the optimal example. Based on this, the phage fertilizer composition targeting tobacco sickle root rot proposed in this invention can be widely used in tobacco planting to prevent and control tobacco sickle root rot and promote tobacco plant growth. Specific application methods include soil application, furrow application, and root irrigation, which can be adapted to different planting scenarios and cultivation modes. It can effectively solve the problem of high incidence of tobacco sickle root rot, improve tobacco yield and quality, and has significant economic and ecological benefits. In the description of this specification, the references to the terms "an embodiment," "example," and "specific example" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples; The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to any specific implementation. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

[0020] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0021] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A phage-based fertilizer composition targeting tobacco sickle root rot, characterized in that, By weight, it includes the following components: 10-30 parts of phage component, 60-85 parts of nutrient component, and 5-15 parts of functional adjuvant. The nutritional components include: 15-25 parts nitrogen source, 10-20 parts phosphorus source, 15-30 parts potassium source, and 5-10 parts trace elements. The nitrogen source is composed of a mixture of compound amino acids and coated lysine in a mass ratio of 3:1, and the compound amino acids are composed of glutamic acid and aspartic acid in a mass ratio of 2:

1. The phosphorus source is potassium dihydrogen phosphate; the potassium source is potassium sulfate. The trace elements are prepared by chelation process of calcium chloride, magnesium sulfate, zinc sulfate and boric acid in a mass ratio of 4:3:2:1 to obtain EDTA-citric acid composite chelate; The functional adjuvants include 2-5 parts of phage protectant, 3-10 parts of soil conditioner, 5-10 parts of carrier and adsorbent, and 0-3 parts of adhesive. The phage protectant is composed of trehalose, skim milk powder, and glycerol in a mass ratio of 2:1:0.

5. The soil conditioner is composed of humic acid, tobacco straw-based biochar, and chitosan in a mass ratio of 3:2:

1. The carrier and adsorbent are attapulgite clay activated with hydrochloric acid; The adhesive is either xanthan gum or soluble starch.

2. The phage fertilizer composition according to claim 1, characterized in that, The method for preparing the phage components is as follows: A1. Collect soil samples from the 0-20cm rhizosphere layer of tobacco fields affected by Fusarium root rot, with each sample weighing 50g. Add the soil samples to LB enrichment medium containing Fusarium oxysporum inoculum, with an initial concentration of 1×10⁻⁶. 6 The culture medium was prepared with CFU / mL, and 0.5% glucose, 0.3% yeast extract, and 0.1% calcium chloride were added. The medium was then incubated at 30℃ and 180 rpm for 24 h with shaking. After centrifugation at 8000 rpm for 20 min, the supernatant was filtered through a 0.22 μm filter membrane to obtain the phage enrichment solution. A2. The phage enrichment solution was separated using the double-layer plate method. The lower layer was LB medium containing 2% agar, and the upper layer was LB medium containing 0.7% agar. The phage enrichment solution was serially diluted and mixed with Fusarium oxysporum bacterial suspension. The mixture was then evenly spread on the upper layer of medium and incubated at 30°C for 12-16 hours. Clear plaques were picked and the purification was repeated 3-5 times to obtain a single phage strain. A3. Through morphological observation, 16S rRNA gene sequencing and fragmentation spectrum determination, two different strains with fragmentation rate ≥90% and strong host specificity were screened out and named FoP1 strain and FoP2 strain, respectively. A4. Using LB medium as a base, a fermentation medium was constructed by adding 0.5% glucose and 0.3% yeast extract. A single phage strain was inoculated and fermented for 18-24 hours at a temperature of 30-32℃, pH 7.0-7.2, aeration rate of 1.0-1.2 vvm, and stirring speed of 200-250 r / min. After fermentation, the phage was concentrated by centrifugation at 8000 r / min for 20 min to obtain a titer of not less than 1×10¹. 0 A single-strain concentrate with PFU / mL was prepared by mixing FoP1 and FoP2 strains at a mass ratio of 1:1-3. The final phage component titer was 5 × 10⁻⁶. 9 -1×10¹ 0 PFU / mL.

3. The phage fertilizer composition according to claim 2, characterized in that, The method for preparing coated lysine from the nitrogen source is as follows: using lysine as the core material, sodium alginate and chitosan at a mass ratio of 2:1 as coating materials, a double-layer coating process is adopted, and coating is carried out at 45-50℃ and stirring speed of 60-80r / min for 2-3 hours to obtain coated lysine with a coating rate of ≥85%.

4. The phage fertilizer composition according to claim 3, characterized in that, The method for preparing the phosphorus source potassium dihydrogen phosphate is as follows: potassium dihydrogen phosphate is purified by recrystallization. Potassium dihydrogen phosphate is dissolved in deionized water at 80-90℃ to prepare a solution with a mass fraction of 30-40%. After stirring and dissolving, the solution is kept at a constant temperature for 4-6 hours. After filtration and cooling for crystallization, the solution is separated by centrifugation and dried at 105℃ for 3 hours to obtain potassium dihydrogen phosphate with a purity ≥99.5% and an impurity content ≤0.1%. The method for preparing potassium sulfate is as follows: potassium sulfate is pulverized to 100-120 mesh, dried at 105℃ for 2 hours to remove moisture, and potassium sulfate with K2O content ≥50% and moisture content ≤0.5% is obtained.

5. The phage fertilizer composition according to claim 4, characterized in that, The method for preparing the trace elements is as follows: Calcium chloride, magnesium sulfate, zinc sulfate, and boric acid are weighed in a mass ratio of 4:3:2:1, dissolved in deionized water, and the total concentration of metal ions is controlled at 0.2 mol / L. EDTA and citric acid are added, and the molar ratio of EDTA to citric acid is controlled at 1:0.

5. The pH is adjusted to 5.5-6.5, and the chelation reaction is carried out at 60-70℃ and a stirring speed of 100-120 r / min for 3-4 hours to obtain the EDTA-citric acid composite chelate.

6. The phage fertilizer composition according to claim 5, characterized in that, The preparation method of the phage protectant is as follows: weigh trehalose, skim milk powder and glycerin in a mass ratio of 2:1:0.5, mix them at 10-15℃ and stirring speed of 50-60 r / min for 20-30 min to obtain a uniform mixture.

7. The phage fertilizer composition according to claim 6, characterized in that, The soil conditioner is prepared as follows: tobacco stalks are crushed to 20-40 mesh and pyrolyzed under anaerobic conditions at 500-600℃ for 3-4 hours to obtain tobacco stalk-based biochar with a specific surface area ≥300m² / g; humic acid, tobacco stalk-based biochar, and chitosan are weighed at a mass ratio of 3:2:1 and mixed at 25-30℃ and a stirring speed of 80-100r / min for 30 minutes to obtain the soil conditioner.

8. The phage fertilizer composition according to claim 7, characterized in that, The carrier is prepared as follows: attapulgite raw material is soaked in a 5% hydrochloric acid solution, with a liquid-to-solid ratio of 5:

1. It is activated at 80℃ and a stirring speed of 60-80 r / min for 2 hours. After activation, it is washed with deionized water until the pH reaches 6.5-7.5, dried at 110℃ for 4 hours, and pulverized to 80-100 mesh to obtain activated attapulgite with a specific surface area ≥150 m² / g and an adsorption capacity ≥80 mg / g.

9. The phage fertilizer composition according to claim 8, characterized in that, The preparation method of the fertilizer-pesticide composition includes the following steps: S1. Preparation of phage-carrier adsorption solution: Take the phage components prepared in steps A1 to A4 and mix them with the phage protectant at a mass ratio of 5:1 to obtain the phage protectant solution; mix the phage protectant solution with activated attapulgite at a mass ratio of 1:2-3, and adsorb for 60-90 min at 15-20℃ and a stirring speed of 80-100 r / min to obtain wet material; S2. Preparation of nutrient substrate: Take 15-25 parts of nitrogen source, 10-20 parts of phosphorus source, 15-30 parts of potassium source, and 5-10 parts of trace elements, and mix them at 30-40℃ and 150-200r / min for 30-40min to obtain the nutrient substrate. S3. Preparation of composite additive: Take 3-10 parts of soil conditioner and 0-3 parts of adhesive, stir at 25-30℃ and 100-120r / min for 20-30min to obtain composite additive; S4. Mixing and granulation: Mix the wet material obtained in step S1, the nutrient base material obtained in step S2, and the composite additive obtained in step S3, add deionized water to make the material moisture content 15-20%, put it into the granulator, adjust the drum speed to 30-40 r / min, the drum inclination angle to 3-5°, and the material residence time to 15-20 min to make granules of 2.8-4.2 mm; S5. Drying and sieving: The particles obtained in step S4 are dried in stages. The first stage is dried at 40°C for 60 minutes, and the second stage is dried at 45°C for 30 minutes. The drying is carried out until the moisture content is ≤5%. After sieving through 2.8 mm and 4.2 mm standard sieves, the phage fertilizer composition is obtained.

10. The application of a phage fertilizer composition targeting tobacco sickle root rot as described in any one of claims 1-9 in the prevention and control of tobacco sickle root rot and the promotion of tobacco plant growth.

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