Antagonistic fungus preparation for sweet potato root rot as well as preparation method and application method of antagonistic fungus preparation

Abstract

The invention relates to the technical field of agricultural microbial preparations, and discloses a sweet potato root rot antagonistic fungus preparation as well as a preparation method and an application method thereof. The preparation consists of a trichoderma harzianum and trichoderma atroviride compound strain, a modified charcoal-humic acid organic carrier, a chitosan-sodium alginate microcapsule wall material, a dextrin-trehalose stabilizer and a sodium salicylate / methyl jasmonate synergistic factor, and is prepared by adopting a solid state fermentation process, a fluidized bed drying process, a microcapsule embedding process and a fluidized bed granulation process. The field control effect reaches 78%-92%, the lasting period is 3-4 months, and the pesticide composition has no inhibition effect on beneficial microorganisms in soil.

Description

Technical Field

[0001] The invention relates to the field of agricultural microbial preparation technology, specifically to a fungal preparation for sweet potato root rot and its preparation and application methods. Background Technology

[0002] Sweet potato (Ipomoea batatas) is the world's seventh largest food crop, with a planting area of ​​approximately 4.5 million hectares in my country and an annual output exceeding 50 million tons. However, the occurrence of sweet potato root rot seriously restricts the healthy development of the sweet potato industry. Sweet potato root rot is a typical soil-borne fungal disease, mainly caused by pathogens such as Fusarium solani f. sp. batatas, Fusarium solani, and Fusarium oxysporum. Infected sweet potatoes exhibit blackening and rotting of the roots, wilting and yellowing of the above-ground parts, and reduced or absent tuber formation. In severe cases, it can lead to yield reductions of 30%-80%, or even total crop failure.

[0003] According to a PubMed database search, Rahman et al. reported the biocontrol efficacy of Trichoderma fungi against Fusarium wilt diseases in Solanaceae crops in Frontiers in Plant Science (2023, 14:1141506, DOI: 10.3389 / fpls.2023.1141506). The study showed that the combined use of Trichoderma fungi reduced the disease incidence by 47.50% and increased yield by 54.49%. Jangir et al. developed a water-dispersible granule based on Bacillus subtilis and Trichoderma harzianum in Scientific Reports (2021, 11: 22895, DOI: 10.1038 / s41598-021-02284-1), which achieved good results in controlling tomato wilt, reducing the disease incidence by 48%. Frontiers in Microbiology (2023, DOI: 10.3389 / fmicb.2023.1160551) reviews the application of Trichoderma fungi in the biocontrol of plant diseases, pointing out that Trichoderma exerts its biocontrol function through multiple mechanisms such as competition, parasitism, antibiosis, and induction of plant resistance.

[0004] Currently, the control of sweet potato root rot mainly relies on chemical pesticides, such as benzimidazole fungicides like carbendazim and thiophanate-methyl. However, the long-term and extensive use of chemical pesticides has led to problems such as increased pathogen resistance, excessive pesticide residues, and imbalances in the soil microbial community. Chinese patent application CN118995429A discloses a strain of Trichoderma echinococcus and its application. This strain achieves a 100% inhibition rate against Fusarium solani, a specific type of sweet potato rot, and its microbial inoculant shows control efficacy of 88.09% against ginger stem rot and 80.20% against sweet potato root rot. However, this technical solution has the following shortcomings: First, it uses a single strain, resulting in a relatively narrow antagonistic spectrum, making it difficult to cope with complex field pathogen communities; second, it uses a wettable powder formulation, which limits the survival and colonization capacity of the inoculant in the soil, resulting in a short duration of effectiveness; third, it does not use microencapsulation technology, making the live bacteria susceptible to inactivation due to environmental stress during storage and application; and fourth, it lacks the synergistic effect of synergistic factors, making it difficult to fully stimulate plant systemic resistance.

[0005] Therefore, developing a biocontrol agent composed of a compound bacterial strain, with slow-release and long-lasting effects, capable of efficiently colonizing the rhizosphere of sweet potato and inducing systemic resistance in plants, is of great practical significance and application value for achieving green control of sweet potato root rot. Summary of the Invention

[0006] The purpose of this invention is to provide an antifungal agent for sweet potato root rot, its preparation method and application method, so as to overcome the shortcomings of existing technologies such as narrow antifungal spectrum of single strains, poor stability of preparations and short duration of effect, and to achieve efficient, long-lasting and environmentally friendly control of sweet potato root rot.

[0007] The first aspect of the present invention provides a fungal antagonistic preparation for sweet potato root rot, comprising the following components and their weight parts: 25-45 parts of a compound antagonistic fungal active ingredient, 30-50 parts of an organic compound carrier, 8-15 parts of a microcapsule wall material, 5-12 parts of a stabilizer, and 2-8 parts of a synergist.

[0008] Furthermore, the composite antagonistic fungal active ingredient is composed of mixed spore powder of *Trichoderma harzianum* and *Trichoderma atroviride*, with a mass ratio of *Trichoderma harzianum* to *Trichoderma atroviride* of 2-4:1. *Trichoderma harzianum* exhibits strong hyperparasitic and competitive activity, directly parasitizing the hyphae of pathogens and secreting cell wall degrading enzymes such as chitinase and β-1,3-glucanase; *Trichoderma atroviride*, on the other hand, possesses strong antimicrobial activity and can produce various antimicrobial metabolites. The synergistic effect of the two significantly enhances the antagonistic effect against Fusarium pathogens. The effective viable count in the composite antagonistic fungal active ingredient is not less than... CFU / g is used to ensure adequate prevention and control.

[0009] Furthermore, the organic composite carrier is composed of modified biochar and humic acid mixed in a mass ratio of 3-5:1. The modified biochar, after alkali activation treatment, possesses a well-developed pore structure and a large specific surface area (350-450 nm). The humic acid ( / g) provides a good colonization site and protective space for antagonistic fungi. Rich in active functional groups such as carboxyl and hydroxyl groups, it can improve soil physicochemical properties, promote the colonization and reproduction of antagonistic fungi in the rhizosphere, and provide nutrients for sweet potato roots.

[0010] Furthermore, the microcapsule wall material is composed of chitosan and sodium alginate in a mass ratio of 1-2:1. Chitosan possesses good biocompatibility, antibacterial activity, and film-forming properties, while sodium alginate can interact with... A stable gel network structure is formed. The chitosan-sodium alginate bilayer microcapsule encapsulation technology effectively protects antagonistic fungal spores from adverse external environments and enables the slow release of live bacteria, thus prolonging the formulation's duration of action.

[0011] Furthermore, the stabilizer is composed of dextrin and trehalose in a mass ratio of 2-3:1. Dextrin, as a commonly used microbial protectant, can form a protective film on the surface of the cells to prevent cell dehydration; trehalose is a non-reducing disaccharide that can replace intracellular water molecules in binding to proteins and phospholipid membranes, maintaining the biological activity and structural integrity of the cells, and improving the storage stability of the preparation.

[0012] Furthermore, the synergistic factor is one or a combination of sodium salicylate or methyl jasmonate. Sodium salicylate is a key signaling molecule for systemically acquired resistance (SAR) in plants, and methyl jasmonate is an important hormone for inducing systemic resistance (ISR). Both can activate the defense response of sweet potato, enhance the host's resistance to pathogens, and form a synergistic effect with the biocontrol effect of antagonistic fungi.

[0013] A second aspect of the present invention provides a method for preparing the above-mentioned antagonistic fungal agent against sweet potato root rot, comprising the following steps:

[0014] Step 1: Preparation of the compound antagonistic fungal active ingredients. *Trichoderma harzianum* and *Trichoderma viride* were inoculated onto PDA agar slant and activated at 25-28℃ for 5-7 days. Seed culture was prepared from the activated strains using potato dextrose broth and cultured at 25-28℃ and 150-180 rpm for 48-72 hours. The seed culture was then inoculated at an inoculation rate of 8%-12% into a solid-state fermentation medium. The solid-state fermentation medium consisted of: 40%-50% corn flour, 30%-40% wheat bran, 10%-20% soybean meal, 1%-3% glucose, and 0.5%-1.5% ammonium sulfate, with an initial moisture content of 55%-65% and an initial pH of 5.5-6.5. The fermentation temperature was controlled at 25-30℃, and the fermentation time was 120-168 hours, with the medium being turned every 24 hours to ensure uniform oxygen supply. After fermentation, the fermentation product is placed in a fluidized bed dryer and dried at an inlet air temperature of 55-65℃ and an outlet air temperature of 35-45℃ until the moisture content is below 8%. The dried fermentation product is then pulverized by a pulverizer, passed through an 80-100 mesh sieve, and mixed at a mass ratio of 2-4:1 to obtain a compound antagonistic fungal active ingredient.

[0015] Step 2: Preparation of the organic composite carrier. Corn stalks or wheat stalks are selected as raw materials, first crushed to particles smaller than 5 mm, and then pyrolyzed in a muffle furnace at 450-550℃ under anaerobic conditions for 2-3 h to prepare biochar. The biochar is mixed with a 10%-20% potassium hydroxide solution at a solid-liquid ratio of 1:5-1:10 (g / mL), and activated by soaking at room temperature for 4-8 h. Then, it is repeatedly washed with deionized water until the pH of the filtrate is close to neutral (pH 6.5-7.5), and dried in an oven at 80-100℃ to constant weight. The modified biochar is then mixed with humic acid at a mass ratio of 3-5:1 to obtain the organic composite carrier.

[0016] Step 3, Microencapsulation. Chitosan (degree of deacetylation 85%-95%, molecular weight 100,000-500,000 Da) is dissolved in a 1%-2% acetic acid solution to prepare a 1.5%-2.5% chitosan solution, and the pH is adjusted to 4.5-5.5. The compound antagonistic fungal active ingredient is suspended in the chitosan solution at a ratio of 1:8-1:12 (g / mL), and stirred until homogeneous to form a spore suspension. A 1%-2% sodium alginate solution and containing... A calcium chloride crosslinking solution with a concentration of 0.5%-1.5% was prepared, and Tween-80 with a mass concentration of 0.1%-0.3% was added as an emulsifier. Using a peristaltic pump, the spore suspension was dripped into the crosslinking solution through a nozzle with an orifice diameter of 0.3-0.5 mm at a flow rate of 2-5 mL / min. During the dripping process, the crosslinking solution was stirred at a speed of 100-200 rpm. After the dripping was completed, stirring was continued for 20-40 minutes to solidify the mixture, allowing chitosan and calcium alginate to form a bilayer microcapsule structure that encapsulates the antagonistic fungal spores. The formed microcapsules were washed 2-3 times with sterile deionized water and drained for later use.

[0017] Step 4, Formulation Preparation. By weight, mix 25-45 parts of microencapsulated antagonistic fungi, 30-50 parts of organic composite carrier, 5-12 parts of stabilizer (dextrin:trehalose = 2-3:1), and 2-8 parts of synergist evenly. Granulate using a fluidized bed granulator at 35-45℃, using deionized water or a 5%-10% dextrin solution as a binder to produce granules with a particle size of 0.5-2.0 mm; or pulverize using an air jet mill to a particle size less than 74 μm to produce a wettable powder. After passing inspection, seal the finished product and store it in a cool, dry place at 4-25℃.

[0018] A third aspect of the present invention provides a method for applying the above-mentioned antagonistic fungal preparation for sweet potato root rot in the prevention and control of sweet potato root rot, comprising one or more of the following application methods:

[0019] Method 1: Seed potato treatment. Prepare a 0.5%-1.5% (w / w) fungal suspension using the antagonistic fungal agent for sweet potato root rot according to this invention. Completely immerse the sweet potato seed potatoes to be planted in the fungal suspension for 10-30 minutes, remove and air-dry, then plant using conventional methods. Seed potato treatment allows the antagonistic fungi to form a protective layer on the seed potato surface, preferentially occupying the rhizosphere niche during sweet potato germination and rooting, effectively preventing pathogen infection.

[0020] Method 2: Trench application. 7-15 days before sweet potato planting, mix 2-5 kg ​​of the sweet potato root rot antagonistic fungal preparation of this invention with 20-30 kg of fine soil or organic fertilizer per acre, and apply evenly to the planting trench, then cover with soil. Trench application allows the antagonistic fungi to establish a dominant microbial community in the soil beforehand, improving the rhizosphere microecological environment and reducing the number and activity of pathogens in the soil.

[0021] Method 3: Root irrigation treatment. During the sweet potato growing season (15-30 days after planting to 30 days before harvest), the sweet potato root rot antagonistic fungal preparation of this invention is formulated to an effective bacterial count of [missing information]. - Apply 100-300 mL of a CFU / mL bacterial suspension per plant, ensuring full penetration into the rhizosphere soil. Depending on field disease incidence, apply once every 15-30 days, for a total of 2-4 applications. This root drenching treatment continuously replenishes antagonistic fungi during critical growth stages of sweet potatoes, maintaining a high concentration of biocontrol bacteria in the rhizosphere and effectively inhibiting the occurrence and spread of pathogens.

[0022] Preferably, the above application methods can be combined to form a comprehensive prevention and control technical solution of "seed potato treatment + furrow application + root irrigation" to further improve the prevention and control effect.

[0023] Furthermore, the antagonistic fungal preparation for sweet potato root rot of the present invention can be used in combination with a reduced dosage of chemical fungicide. The chemical fungicide is one of carbendazim, thiophanate-methyl, or fludioxonil, and the dosage is 30%-50% of the conventional dosage. Through bio-chemical synergy, the dosage of chemical pesticides can be reduced by 50%-70% while ensuring control efficacy, thereby reducing environmental pollution and pesticide residue risks.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] First, this invention employs a compound formulation of *Trichoderma harzianum* and *Trichoderma viride*. These two antagonistic fungi complement and synergistically enhance each other's biocontrol mechanisms. *Trichoderma harzianum* primarily exerts its parasitic and competitive effects, while *Trichoderma viride* primarily exerts its antibiotic effects. The compound strain exhibits a broader antagonistic spectrum and more stable biocontrol efficacy. Experimental results show that the compound strain achieved plate inhibition rates of 95.6%, 89.3%, and 92.1% against *Fusarium solani*, *Fusarium solani*, and *Fusarium oxysporum*, respectively, significantly higher than that of single strains.

[0026] Secondly, this invention employs a chitosan-sodium alginate double-layer microcapsule encapsulation technology, effectively protecting antagonistic fungal spores from adverse environmental factors such as dryness, ultraviolet radiation, and high temperatures. Microencapsulation significantly improves the formulation's stability during room temperature storage; after 12 months of storage at 4-25℃, the viable bacterial count remains at [value missing]. With a CFU / g or higher, the survival rate is greater than 80%. At the same time, the sustained-release properties of the microcapsules allow the antagonistic fungi to be continuously released in the soil, with an effective period of 3-4 months, which is far superior to the 1-2 months of conventional wettable powders.

[0027] Third, this invention uses a composite carrier of modified biochar and humic acid. The porous structure of biochar provides an ideal colonization microenvironment for antagonistic fungi, while humic acid improves the soil's physicochemical properties and microbial community structure. Pot experiments and field trials show that after applying the formulation of this invention, the number of Trichoderma in the rhizosphere soil of sweet potatoes increased by 15-25 times compared to the control, the number of pathogens decreased by 60%-85%, and the soil microbial diversity index increased by 20%-35%.

[0028] Fourth, the addition of sodium salicylate or methyl jasmonate as synergistic factors in this invention can activate the systemic resistance response of sweet potato, upregulate the expression of defense-related genes (such as PR-1, PR-2, PR-5, PDF1.2, etc.), and increase the activity of defense enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO). The synergistic effect of plant resistance induction and direct biocontrol against antagonistic fungi further enhances the disease control efficacy of the formulation.

[0029] Fifth, field plot trials showed that the antagonistic fungal preparation for sweet potato root rot of this invention achieved a control effect of 78%-92% against sweet potato root rot, which is 8%-12% higher than the Trichoderma hygroscopicum HM-8 preparation disclosed in comparative document CN118995429A (control efficacy of approximately 80%). Sweet potato yields treated with this preparation increased by 45%-68% compared to the control group and by 12%-25% compared to the chemical pesticide control. Furthermore, this preparation has no inhibitory effect on beneficial microorganisms in the soil (such as actinomycetes and nitrogen-fixing bacteria), exhibits high safety, and meets the requirements of green agricultural development. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be considered as limiting the scope of protection of the present invention.

[0031] The *Trichoderma harzianum* used in this invention embodiment was *Trichoderma harzianum* ACCC 30371, purchased from the China Agricultural Microbiological Culture Collection Center; the *Trichoderma viride* was *Trichoderma viride* ACCC 30369, purchased from the China Agricultural Microbiological Culture Collection Center. *Fusarium solani* f. sp. batatas, *Fusarium solani*, and *Fusarium oxysporum*, among other pathogens, were provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Chitosan (degree of deacetylation 90%, molecular weight 300,000 Da) was purchased from Sinopharm Chemical Reagent Co., Ltd.; sodium alginate (viscosity 300 mPa·s) was purchased from Qingdao Mingyue Algae Group; humic acid (organic matter content ≥60%, humic acid content ≥50%) was purchased from Shandong Quanlin Jiayou Fertilizer Co., Ltd.; sodium salicylate and methyl jasmonate were both analytical grade and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0032] Example 1

[0033] Prepare a sweet potato root rot antagonistic fungal preparation (parts by weight) according to the following formula: 25 parts of compound antagonistic fungal active ingredients, 50 parts of organic compound carrier, 8 parts of microcapsule wall material, 12 parts of stabilizer, and 5 parts of synergist.

[0034] Preparation of the compound antagonistic fungal active ingredients: *Trichoderma harzianum* ACCC 30371 and *Trichoderma viride* ACCC 30369 were inoculated onto PDA medium slant and activated at 26℃ for 6 days. Seed culture was prepared from the activated strains using potato dextrose liquid medium (200 g / L potato extract, 20 g / L glucose), and cultured at 26℃ and 160 rpm for 60 h with shaking. The seed culture was inoculated at a 10% inoculation rate into a solid-state fermentation medium composed of: 45% corn flour, 35% wheat bran, 15% soybean meal, 2% glucose, 1% ammonium sulfate, and the remainder water, with an initial moisture content of 60% and an initial pH of 6.0. Fermentation was controlled at 26℃ for 144 h, with the medium being turned over every 24 h. After fermentation, the fermentation product was placed in a fluidized bed dryer and dried to a moisture content of 6.5% under conditions of 60℃ inlet air temperature and 40℃ outlet air temperature. The dried fermentation products were pulverized, passed through a 100-mesh sieve, and mixed at a mass ratio of 3:1 to obtain a compound antagonistic fungal active ingredient with an effective viable count of [missing information]. CFU / g.

[0035] Preparation of the organic composite carrier: Corn stalks were crushed to a particle size of less than 5 mm and pyrolyzed in a muffle furnace at 500℃ under anaerobic conditions for 2.5 h to prepare biochar. The biochar was mixed with a 15% potassium hydroxide solution at a solid-liquid ratio of 1:8 (g / mL), activated by soaking at room temperature for 6 h, washed with deionized water until pH 7.0, and dried at 90℃. The modified biochar had a specific surface area of ​​385. / g, pore volume is 0.32 / g, pH 8.0. Modified biochar and humic acid were mixed evenly at a mass ratio of 4:1 to obtain an organic composite carrier.

[0036] Microencapsulation: Chitosan was dissolved in a 1.5% (w / w) acetic acid solution to prepare a 2.0% (w / w) chitosan solution, and the pH was adjusted to 5.0. The compound antifungal active ingredient was suspended in the chitosan solution at a ratio of 1:10 (g / mL) and stirred until homogeneous. A 1.5% (w / w) sodium alginate solution and containing... A 1.0% calcium chloride crosslinking solution was prepared, and 0.2% Tween-80 was added to the crosslinking solution. The spore suspension was dripped into the crosslinking solution at a flow rate of 3 mL / min through a 0.4 mm orifice nozzle using a peristaltic pump. The crosslinking solution was stirred at 150 rpm during the dripping process. After solidification for 30 min, the solution was washed three times with sterile deionized water and drained.

[0037] Formulation: Microencapsulated antagonistic fungi were mixed evenly with an organic composite carrier, stabilizer (dextrin:trehalose = 2.5:1), and sodium salicylate. Granulation was performed using a fluidized bed granulator at 40℃, using an 8% (w / w) dextrin solution as a binder to produce granules with a particle size of 0.8-1.5 mm. The effective viable count of the finished product was... CFU / g.

[0038] Example 2

[0039] Prepare a sweet potato root rot antagonistic fungal preparation (parts by weight) according to the following formula: 35 parts of compound antagonistic fungal active ingredients, 40 parts of organic compound carrier, 12 parts of microcapsule wall material, 8 parts of stabilizer, and 5 parts of synergist.

[0040] Preparation of the compound antagonistic fungal active ingredient: Prepared according to the method in Example 1, but the mass ratio of *Trichoderma harzianum* to *Trichoderma viride* was adjusted to 2.5:1. The composition of the solid-state fermentation medium was adjusted to: 50% corn flour, 30% wheat bran, 12% soybean meal, 3% glucose, and 1.2% ammonium sulfate. The initial moisture content was 58%, the initial pH was 5.8, the fermentation temperature was 28℃, and the fermentation time was 156 h. The effective viable cell count after drying was... CFU / g.

[0041] Preparation of the organic composite carrier: Prepared according to the method in Example 1, but with the mass ratio of modified biochar to humic acid adjusted to 3.5:1, the alkali activation time to 5 h, and the specific surface area of ​​the modified biochar to 410. / g, pore volume is 0.35 / g, pH 8.2.

[0042] Microencapsulation: Performed according to the method in Example 1, but the concentration of chitosan solution was adjusted to 2.2%, the concentration of sodium alginate solution was adjusted to 1.8%, and the calcium chloride crosslinking solution was adjusted accordingly. The concentration was adjusted to 1.2%, and the mass ratio of chitosan to sodium alginate was 1.5:1.

[0043] Formulation: Microencapsulated antagonistic fungi were mixed evenly with an organic composite carrier, stabilizer (dextrin:trehalose = 2:1), and synergist (sodium salicylate: methyl jasmonate = 1:1). Granulation was performed using a fluidized bed granulator at 38℃ to produce granules with a particle size of 1.0-1.8 mm. The effective viable count of the finished product was [missing information]. CFU / g.

[0044] Example 3

[0045] Prepare a sweet potato root rot antagonistic fungal preparation (parts by weight) according to the following formula: 45 parts of compound antagonistic fungal active ingredients, 30 parts of organic compound carrier, 15 parts of microcapsule wall material, 5 parts of stabilizer, and 5 parts of synergist.

[0046] Preparation of the compound antagonistic fungal active ingredient: Prepared according to the method in Example 1, but the mass ratio of *Trichoderma harzianum* to *Trichoderma viride* was adjusted to 4:1. The composition of the solid-state fermentation medium was adjusted to: corn flour 40%, wheat bran 40%, soybean meal 15%, glucose 1.5%, ammonium sulfate 0.8%, initial moisture content 62%, initial pH 6.2, fermentation temperature 25℃, and fermentation time 168 h. The effective viable count after drying was... CFU / g.

[0047] Preparation of the organic composite carrier: Prepared according to the method in Example 1, but with the mass ratio of modified biochar to humic acid adjusted to 5:1, the pyrolysis temperature set at 550℃, the alkali activation time at 4 h, and the specific surface area of ​​the modified biochar at 435 nm. / g, pore volume is 0.38 / g, pH 7.8.

[0048] Microencapsulation: Performed according to the method in Example 1, but the concentration of chitosan solution was adjusted to 2.5%, the concentration of sodium alginate solution was adjusted to 2.0%, and the calcium chloride crosslinking solution was... The concentration was adjusted to 1.5%, and the mass ratio of chitosan to sodium alginate was 2:1.

[0049] Formulation: Microencapsulated antagonistic fungi were mixed thoroughly with an organic composite carrier, stabilizer (dextrin:trehalose = 3:1), and methyl jasmonate. The mixture was then pulverized using an air jet mill to a particle size of less than 74 μm to prepare a wettable powder. The effective viable count of the finished product was [missing information]. CFU / g.

[0050] Comparative Example 1 (single strain, without microencapsulation)

[0051] A fungal antagonist preparation for sweet potato root rot was prepared according to the following formula (parts by weight): 35 parts *Trichoderma harzianum* spore powder, 50 parts soluble starch, and 15 parts dextrin. *Trichoderma harzianum* ACCC 30371 was subjected to solid-state fermentation, drying, and pulverization according to the method in Example 1. It was then directly mixed with soluble starch and dextrin without microencapsulation to prepare a wettable powder. The effective viable count of the finished product was... CFU / g.

[0052] Comparative Example 2 (single strain, with microencapsulation)

[0053] The following formula (parts by weight) was used to prepare an antagonistic fungal preparation for sweet potato root rot: 35 parts *Trichoderma harzianum* spore powder, 40 parts organic composite carrier, 12 parts microcapsule wall material, 8 parts stabilizer, and 5 parts synergist. *Trichoderma harzianum* ACCC 30371 was cultured, microencapsulated, and prepared according to the method in Example 2, but without adding *Trichoderma viride*. The effective viable count of the finished product was... CFU / g.

[0054] Comparative Example 3 (compound strain, without microencapsulation, without synergistic factors)

[0055] The following formula (parts by weight) was used to prepare an antagonistic fungal preparation for sweet potato root rot: 35 parts of compound antagonistic fungal active ingredients, 40 parts of organic compound carrier, 15 parts of dextrin, and 10 parts of soluble starch. *Trichoderma harzianum* and *Trichoderma viride* were cultured and mixed according to the method in Example 2, but without microencapsulation and without adding synergistic factors. The effective viable count of the finished product was... CFU / g.

[0056] Test Example 1: Plate Antibacterial Test

[0057] The antagonistic effect of the formulations in each example and comparative example against the main pathogen of sweet potato root rot was determined using the plate confrontation method. The formulations were prepared to have an effective bacterial count of [missing information]. A bacterial suspension of CFU / mL was prepared. A 5 mm diameter bacterial pellet was inoculated into the center of a PDA plate, and the bacterial suspension was spread on the edge of the plate. After incubation at 28°C for 5 days, the diameter of the bacterial colony was measured, and the inhibition rate was calculated.

[0058] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100%

[0059] The experimental results are shown in Table 1.

[0060] Table 1. Plate inhibition rate (%) of each formulation of the examples and comparative examples against the pathogen of sweet potato root rot

[0061] sample Solanum Fusarium wilt-specific sweet potato strain Fusarium solani Fusarium oxysporum Example 1 93.2±2.1 86.5±1.8 89.4±2.3 Example 2 95.6±1.5 89.3±2.0 92.1±1.6 Example 3 94.8±1.9 88.1±2.2 90.8±2.0 Comparative Example 1 78.4±2.6 72.3±2.5 75.6±2.8 Comparative Example 2 85.2±2.3 79.6±2.1 82.4±2.4 Comparative Example 3 88.6±2.0 82.5±1.9 85.3±2.1

[0062] Note: Data are the mean ± standard deviation of three repeated trials.

[0063] Test results showed that the inhibitory rates of the formulations in Examples 1-3 against the three main pathogens were higher than those in Comparative Examples 1-3. Among them, the formulation in Example 2 showed the best inhibitory effect, with inhibition rates of 95.6%, 89.3%, and 92.1% against Fusarium solani (a specific strain of Fusarium solani), Fusarium solani (a specific strain of Fusarium solani), and Fusarium oxysporum (a specific strain of Fusarium solani). The inhibitory rate of the compound strain (Example 2) was 10%-12% higher than that of the single strain (Comparative Example 2), demonstrating the synergistic effect of Trichoderma harzianum and Trichoderma viride.

[0064] Test Example 2: Storage Stability Test

[0065] The formulations of each example and comparative example were stored in the dark at 4°C, 15°C and 25°C, respectively, and samples were taken periodically to detect the number of effective viable bacteria and calculate the survival rate.

[0066] Survival rate (%) = (Number of viable bacteria after storage / Initial number of viable bacteria) × 100%

[0067] The experimental results are shown in Table 2.

[0068] Table 2. Viable bacterial survival rate (%) of each example and comparative formulation under storage conditions at 25°C.

[0069] sample 1 month 3 months 6 months 9 months 12 months Example 1 95.2 88.6 82.4 76.5 72.3 Example 2 96.8 91.2 85.6 80.2 76.8 Example 3 97.5 92.8 87.3 82.6 78.5 Comparative Example 1 82.3 65.4 48.2 35.6 25.4 Comparative Example 2 94.5 86.3 78.5 70.2 64.3 Comparative Example 3 85.6 72.5 58.6 46.8 38.2

[0070] Test results showed that the storage stability of the formulations of Examples 1-3 and Comparative Example 2, which used microencapsulation technology, was significantly better than that of Comparative Examples 1 and 3, which did not undergo microencapsulation. After 12 months of storage at 25°C, the viable bacterial survival rate of the formulations of Examples 1-3 was 72.3%-78.5%, while the survival rates of Comparative Examples 1 and 3 were only 25.4% and 38.2%, respectively. This indicates that chitosan-sodium alginate bilayer microcapsules have a good protective effect against antagonistic fungal spores and can significantly extend the shelf life of the formulations.

[0071] Test Example 3: Potted Plant Control Efficacy Test

[0072] The experiment was conducted in a greenhouse. Diseased soil samples were taken (containing *Fusarium solani* var. *sweet potato*, with a pathogen concentration of approximately [missing information]). Mix CFU / g soil with sterilized nutrient soil at a 1:3 ratio, and fill 5 kg of soil into 25 cm diameter flowerpots. Select healthy sweet potato seed tubers (variety: Longshu No. 9) and conduct experiments according to the following treatments:

[0073] Treatment 1 (Formulation of Example 2): The formulation of Example 2 was mixed with the potting soil at a dosage of 10 g per pot and then the seed potatoes were planted.

[0074] Treatment 2 (Comparative Example 1 formulation): Mix the Comparative Example 1 formulation at a dosage of 10 g per pot with the potting soil and then plant the seed potatoes.

[0075] Treatment 3 (Comparative Example 2 formulation): Mix the Comparative Example 2 formulation at a dosage of 10 g per pot with the potting soil and then plant the seed potatoes.

[0076] Treatment 4 (chemical pesticide control): Seed potatoes were soaked in a 500-fold dilution of 50% carbendazim wettable powder for 30 minutes before planting.

[0077] Treatment 5 (blank control): No treatment was performed, and seed potatoes were directly planted in the diseased soil.

[0078] Each treatment was replicated three times, with 10 pots per replicate. Routine management was implemented. Disease incidence was assessed 60 days after planting, and the disease index and control efficacy were calculated.

[0079] Disease severity grading criteria: Grade 0: Healthy plant with no symptoms; Grade 1: Roots have a small number of lesions (<10%), above-ground parts are normal; Grade 2: Roots have lesions accounting for 10%-30%, above-ground parts are slightly wilted; Grade 3: Roots have lesions accounting for 30%-60%, above-ground parts are obviously wilted and yellowed; Grade 4: Roots have lesions accounting for more than 60% or roots are rotten, the plant is near death or dead.

[0080] Disease index = [∑(number of diseased plants at each level × corresponding level value) / (total number of plants surveyed × highest level value)] × 100;

[0081] Prevention and control effect (%) = [(disease index of blank control - disease index of treatment) / disease index of blank control] × 100;

[0082] The experimental results are shown in Table 3.

[0083] Table 3. Control effect of pot experiment

[0084] deal with Incidence rate (%) Disease index Prevention and control efficacy (%) Process 1 (Example 2) 16.7±3.5 8.5±1.8 85.2±2.1 Treatment 2 (Comparative Example 1) 43.3±5.2 25.6±3.2 55.4±3.8 Treatment 3 (Comparative Example 2) 26.7±4.1 15.2±2.5 73.5±2.9 Treatment 4 (Chemical pesticides) 30.0±4.5 18.5±2.8 67.8±3.2 Treatment 5 (Blank Control) 76.7±6.3 57.4±4.5 -

[0085] Note: Data are the mean ± standard deviation of 3 replicates.

[0086] Pot experiment results showed that the formulation of Example 2 had the best control effect, reaching 85.2%, which was significantly higher than that of Comparative Example 1 (55.4%), Comparative Example 2 (73.5%), and the chemical pesticide control (67.8%). This indicates that the synergistic effect of the compound strain, microencapsulation, and synergistic factors of the present invention can significantly improve the control effect of sweet potato root rot.

[0087] Test Example 4: Field Plot Experiment

[0088] The experimental site was located in Lulong County, Qinhuangdao City, Hebei Province, a major sweet potato producing area. Sweet potatoes had been continuously planted in this area for eight years, and root rot was a serious problem, with an incidence rate of 40%-60% in the past three years. A randomized block design was used, with three replicates per treatment and each plot measuring 50 mu (approximately 3.3 hectares). (5 m × 10 m), row spacing 0.8 m, plant spacing 0.25 m. The tested sweet potato variety was Longshu No. 9.

[0089] The experiment was designed with the following 6 treatments:

[0090] T1: Formulation of Example 1, apply 3 kg / mu in furrows + drench the roots twice (each time diluted 500 times, 150 mL per plant).

[0091] T2: Formulation of Example 2, apply 4 kg / mu in furrows + drench the roots twice (each time diluted 500 times, 150 mL per plant).

[0092] T3: Formulation of Example 3, apply 5 kg / mu in furrows + drench the roots twice (each time diluted 500 times, 150 mL per plant).

[0093] T4: Comparative Formulation 1, apply 4 kg / acre in furrows + drench the roots twice (each time diluted 500 times, 150 mL per plant).

[0094] T5: Chemical pesticide control, 50% carbendazim wettable powder 4 kg / mu mixed with soil and applied in furrows + root drenching once 40 days after planting (500 times dilution).

[0095] T6: Blank control, no medication was administered.

[0096] The experiment was conducted on May 10th. Furrow application was performed 7 days prior to planting, followed by root irrigation treatments 30 and 60 days after planting. Disease incidence and yield were assessed at harvest time on October 15th, and the disease index and control efficacy were calculated using the same method as in test case 3.

[0097] The experimental results are shown in Table 4.

[0098] Table 4. Control efficacy and yield in field plot trials

[0099] deal with Incidence rate (%) Disease index Prevention and control efficacy (%) Yield (kg / mu) Production increase rate (%) T1 18.5±2.8 10.2±1.5 78.3±2.4 2856±125 52.8±3.6 T2 12.3±2.1 6.5±1.2 86.2±1.8 3142±138 68.1±4.2 T3 10.8±1.9 5.8±1.0 87.7±1.6 3086±142 65.1±4.5 T4 35.6±3.8 22.4±2.5 52.3±3.2 2385±118 27.6±3.8 T5 28.2±3.2 16.8±2.1 64.3±2.8 2568±126 37.4±4.0 T6 62.5±4.5 47.0±3.8 - 1869±105 -

[0100] Note: Data are the mean ± standard deviation of 3 replicates.

[0101] Field trial results showed that the control efficacy of the formulations in Examples 1-3 against sweet potato root rot was 78.3%-87.7%, significantly higher than that of the formulation in Comparative Example 1 (52.3%) and the chemical pesticide control (64.3%). Examples 2 and 3 showed the best control efficacy, reaching 86.2% and 87.7%, respectively. Sweet potato yields treated with the formulations in Examples 1-3 increased by 52.8%-68.1% compared to the control group, and by 11.3%-22.4% compared to the chemical pesticide control.

[0102] Test Example 5: Determination of the Duration of Effect of the Formulation

[0103] Based on the field trial in Test Example 4, rhizosphere soil samples were collected at 30, 60, 90, and 120 days after planting. The number of Trichoderma and Fusarium in the soil was determined by the dilution plate method to evaluate the persistence of the formulation.

[0104] The experimental results are shown in Table 5.

[0105] Table 5. Dynamic changes in the number of Trichoderma and Fusarium species in rhizosphere soil under different treatments (×) CFU / g dry soil)

[0106] deal with index 30 days 60 days 90 days 120 days T2 Trichoderma 85.6±6.2 125.3±8.5 98.4±7.1 68.2±5.3 T2 Fusarium 12.3±1.5 8.6±1.2 10.5±1.4 15.8±1.8 T4 Trichoderma 65.2±5.5 42.8±4.2 18.6±2.5 8.5±1.2 T4 Fusarium 18.5±2.1 25.6±2.8 35.2±3.5 48.6±4.2 T6 Trichoderma 3.2±0.5 2.8±0.4 3.5±0.6 3.0±0.5 T6 Fusarium 52.4±4.5 68.5±5.8 85.6±6.5 102.3±8.2

[0107] Test results showed that the number of Trichoderma species in the rhizosphere soil treated with formulation 2 (T2) reached its peak 60 days after planting. (CFU / g), which remained at 120 days. The CFU / g concentration was 22.7 times that of the blank control. Meanwhile, the Trichoderma count treated with formulation 1 (T4) decreased sharply after 60 days, reaching only [amount missing] at 120 days. CFU / g. Meanwhile, the number of Fusarium spores in the T2 treatment remained consistently at a low level ( - (CFU / g), while the Fusarium count in the T6 blank control continued to rise to CFU / g. This indicates that the microencapsulation sustained-release technology of the formulation of this invention can effectively prolong the colonization time of antagonistic fungi in the soil, with a duration of effect of 3-4 months.

[0108] Test Example 6: Analysis of Soil Microbial Diversity

[0109] At the end of the field trial in Test Example 4 (150 days after planting), rhizosphere soil samples were collected from each treatment. Soil bacterial and fungal community structure was analyzed using 16S rRNA and ITS high-throughput sequencing technology, and α diversity indices (Shannon index, Simpson index, Chao1 index) were calculated.

[0110] The experimental results are shown in Table 6.

[0111] Table 6 Soil microbial α-diversity index under different treatments

[0112] deal with Shannon bacteria bacteria Chao1 Shannon fungus Chao1 fungus T2 6.85±0.15 2856±125 4.52±0.12 685±35 T4 6.42±0.18 2485±118 4.15±0.15 612±32 T5 5.86±0.22 2125±102 3.68±0.18 485±28 T6 5.95±0.20 2265±110 3.82±0.16 525±30

[0113] Test results showed that the soil bacterial and fungal diversity indices of the formulation in Example 2 (T2) were higher than those of other treatments, with a bacterial Shannon index of 6.85 and a fungal Shannon index of 4.52, representing increases of 15.1% and 18.3% respectively compared to the blank control (T6). The chemical pesticide control (T5) had the lowest microbial diversity index, indicating that chemical pesticides have a certain inhibitory effect on soil microorganisms. This invention's formulation not only effectively controls sweet potato root rot but also improves the soil microecological environment and enhances soil health.

[0114] Test Example 7: Security Evaluation

[0115] The safety of the formulation in Example 2 was evaluated, including its safety to sweet potatoes and its impact on beneficial soil microorganisms.

[0116] Sweet potato safety test: The formulation of Example 2 was applied to the potting soil at a dosage of 20 g per pot (twice the recommended dosage), and the growth of sweet potatoes was observed. The results showed that the high-dose application of the formulation of this invention had no adverse effects on the growth of sweet potatoes, no phytotoxicity symptoms were observed, and there was no significant difference between the above-ground fresh weight and the underground tuber weight of sweet potatoes and the normal application amount (P>0.05).

[0117] Effects on Beneficial Microorganisms: The plate confrontation method was used to determine the effects of the formulation of Example 2 on beneficial soil microorganisms. The tested beneficial microorganisms included Bacillus subtilis, Bacillus amyloliquefaciens, Paenibacillus polymyxa, and Streptomyces p. The results showed that the inhibition rate of the formulation of the present invention against the above-mentioned beneficial microorganisms was less than 5%, and there was no significant difference compared with the blank control, indicating that the formulation of the present invention is safe for beneficial soil microorganisms.

[0118] In summary, the antagonistic fungal preparation for sweet potato root rot of the present invention has the advantages of strong antagonistic activity, good storage stability, long duration of effect, excellent control effect and high safety. It can effectively replace chemical pesticides for green control of sweet potato root rot and has good application prospects.

[0119] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fungicide for antagonizing sweet potato root rot, characterized in that, It comprises the following components and their weight proportions: 25-45 parts of a compound antagonistic fungal active ingredient, 30-50 parts of an organic composite carrier, 8-15 parts of a microcapsule wall material, 5-12 parts of a stabilizer, and 2-8 parts of a synergist; wherein, the compound antagonistic fungal active ingredient is composed of mixed spore powder of *Trichoderma harzianum* and *Trichoderma viride*, with a mass ratio of *Trichoderma harzianum* to *Trichoderma viride* of 2-4:1, and the effective viable count in the compound antagonistic fungal active ingredient is not less than 5 × 10⁻⁶. 9 CFU / g; the organic composite carrier is composed of modified biochar and humic acid mixed in a mass ratio of 3-5:1; the microcapsule wall material is composed of chitosan and sodium alginate in a mass ratio of 1-2:1; the stabilizer is composed of dextrin and trehalose in a mass ratio of 2-3:1; the synergist is one or a combination of two of sodium salicylate or methyl jasmonate.

2. The sweet potato root rot antagonistic fungal preparation according to claim 1, characterized in that, The *Trichoderma harzianum* is either *Trichoderma harzianum* ACCC 30371 or *Trichoderma harzianum* CGMCC 3.5163, and the *Trichoderma viride* is either *Trichoderma viride* ACCC 30369 or *Trichoderma viride* CGMCC 3.3711.

3. The sweet potato root rot antagonistic fungal preparation according to claim 1, characterized in that, The modified biochar is straw biochar that has undergone alkali activation treatment, with a specific surface area of ​​350-450 m². 2 / g, pore volume 0.25-0.40 cm³ 3 / g, pH 7.5-8.

5.

4. The sweet potato root rot antagonistic fungal preparation according to claim 1, characterized in that, The degree of deacetylation of the chitosan is 85%-95%, and the molecular weight is 100,000-500,000 Da; the viscosity of the sodium alginate is 200-400 mPa·s.

5. The method for preparing the sweet potato root rot antagonistic fungal agent according to any one of claims 1-4, characterized in that, Includes the following steps: Step 1, Preparation of the compound antagonistic fungal active ingredients: Trichoderma harzianum and Trichoderma viride were inoculated into PDA medium slant for activation culture. After preparing seed liquid, it was inoculated into solid fermentation medium at an inoculation rate of 8%-12% for fermentation culture. The fermentation temperature was 25-30℃ and the fermentation time was 120-168 h. The fermentation product was dried in a fluidized bed until the moisture content was less than 8%, pulverized and passed through an 80-100 mesh sieve, and mixed at a mass ratio of 2-4:1 to obtain the compound antagonistic fungal active ingredients. Step 2, preparation of organic composite carrier: After crushing the straw, it is pyrolyzed at 450-550℃ under anaerobic conditions to prepare biochar. Then, it is soaked and activated in a 10%-20% potassium hydroxide solution for 4-8 hours, washed until neutral, dried at 80-100℃, and mixed with humic acid at a mass ratio of 3-5:1 to obtain organic composite carrier. Step 3, Microencapsulation: Chitosan is dissolved in a 1%-2% acetic acid solution to prepare a 1.5%-2.5% chitosan solution. The compound antifungal active ingredient is suspended in the chitosan solution. A 1%-2% sodium alginate solution and a solution containing Ca are then added dropwise via spraying. 2+ Cross-linking and curing were carried out in a calcium chloride cross-linking solution with a concentration of 0.5%-1.5% for 20-40 minutes to form microencapsulated antagonistic fungi. Step 4, Formulation preparation: Mix the microencapsulated antagonistic fungi with the organic composite carrier, stabilizer, and synergist according to the specified ratio, and then use a fluidized bed granulation process to prepare granules or wettable powders. The granulation temperature is 35-45℃, and the particle size is 0.5-2.0 mm.

6. The preparation method according to claim 5, characterized in that, The solid fermentation medium is composed of: 40%-50% corn flour, 30%-40% wheat bran, 10%-20% soybean meal, 1%-3% glucose, and 0.5%-1.5% ammonium sulfate, with an initial moisture content of 55%-65% and an initial pH of 5.5-6.

5.

7. The preparation method according to claim 5, characterized in that, The crosslinking liquid described in step three also contains Tween-80 at a mass concentration of 0.1%-0.3% as an emulsifier.

8. The method of applying the sweet potato root rot antagonistic fungal preparation according to any one of claims 1-4 in the prevention and control of sweet potato root rot, characterized in that, This includes one or more of the following methods of application: Method 1, Seed potato treatment: Prepare a fungal suspension with a mass concentration of 0.5%-1.5% using the aforementioned antagonistic fungal preparation for sweet potato root rot, soak the sweet potato seed potatoes for 10-30 minutes, and then dry them before planting. Method 2, trench application: Before planting sweet potatoes, mix the sweet potato root rot antagonistic fungal preparation at a rate of 2-5 kg ​​per acre with 20-30 kg of fine soil and then spread it in the planting trench. Method 3, root irrigation treatment: During the sweet potato growing season, the aforementioned sweet potato root rot antagonistic fungal preparation is formulated to an effective bacterial count of 1×10⁻⁶. 7 -5×10 8 Apply 100-300 mL of bacterial suspension (CFU / mL) to each plant, once every 15-30 days, for a total of 2-4 applications.

9. The application method according to claim 8, characterized in that, The application period for the method described is from 7-15 days before sweet potato planting to 30 days before harvest.

10. The application method according to claim 8, characterized in that, The application method can be used in conjunction with a reduced dosage of chemical fungicide, wherein the chemical fungicide is one of carbendazim, thiophanate-methyl, or fludioxonil, and the dosage of the chemical fungicide is 30%-50% of the conventional dosage.