Preparation method of carbon chain enzymatic hydrolysate for preventing and treating plant phytophthora root rot

Abstract

The invention discloses a preparation method of carbon chain enzymatic hydrolysate for preventing and treating plant phytophthora root rot, and belongs to the technical field of agricultural biology. According to the preparation method of the carbon chain enzymatic hydrolysate for preventing and treating plant phytophthora root rot, the carbon chain enzymatic hydrolysate is prepared from water, polysaccharide, mineral source potassium fulvate and cellulase according to the weight ratio of 100: (3-6): (6-10): (1-4). The preparation method comprises the following steps: heating water to 100 DEG C to dissolve polysaccharide, cooling to 40 DEG C, adding the mineral source potassium fulvate, mixing, adding cellulase at the temperature not lower than 30 DEG C, carrying out constant-temperature enzymolysis, and finally standing and curing. Through the synergistic effect of all the components, the composition can effectively stimulate plant system resistance, interfere phytophthora and promote plant wound healing, has remarkable prevention and treatment and diseased plant rehabilitation effects on phytophthora root rot such as durian canker and citrus brown rot, and meanwhile solves the problems of pesticide residues, drug resistance and environmental pollution caused by chemical prevention and treatment.

Description

Technical Field

[0001] This invention relates to a method for preparing a carbon-chain enzymatic hydrolysate for controlling Phytophthora blight in plants, belonging to the field of agricultural biotechnology, specifically the direction of plant disease biological control technology. The method uses water, polysaccharides, mineral-derived potassium humate, and cellulase as main raw materials, and prepares the carbon-chain enzymatic hydrolysate for controlling Phytophthora blight through specific ratios and processes. This invention is mainly applicable to the control of Phytophthora blight in various crops such as fruit trees, vegetables, and forest trees, and is particularly effective for the control and rehabilitation of diseases in economic crops such as durian canker, citrus brown rot, and potato late blight. Background Technology

[0002] Phytophthora is one of the most devastating pathogens in agricultural production, causing root rot, stem rot, canker, and fruit rot in various crops, such as durian canker, citrus brown rot, and potato late blight. Conventional control methods mainly rely on chemical fungicides (such as metalaxyl and cymoxanil) and phosphonate injections, but these methods carry risks of environmental pollution, pesticide residue exceeding limits, and the development of drug resistance in pathogens. For example, the detection rate of metalaxyl-resistant strains of Phytophthora has reached as high as 78% (data from the National Agricultural Technology Extension Service Center in 2024); phosphonate residues in fruits such as citrus and durian exceed the 0.05 mg / kg standard stipulated in GB2763-2024 "National Food Safety Standard for Maximum Residue Limits of Pesticides in Food," posing a food safety hazard; at the same time, the extensive use of chemical agents leads to a decline in soil microbial diversity and may pollute groundwater through leaching.

[0003] Physical control methods (such as cleaning up diseased plant debris and soil disinfection) are generally only suitable for small-scale plantations, have high operating costs, and have limited effectiveness on perennial woody plants. In terms of biological control, existing technologies mostly use microbial agents such as Bacillus and Trichoderma, but these suffer from slow onset of action, significant dependence on environmental conditions (such as soil temperature and humidity) on control efficacy, and insufficient stability.

[0004] Humic acids are widely used in agriculture due to their ability to improve soil structure and promote plant growth. However, existing humic acid products and technologies have significant gaps in their application for controlling Phytophthora blight: 1) Formulation gaps: Most existing products are single-component or have unclear mixing ratios (e.g., only labeled "humic acid ≥ 3%)", lacking specific synergistic formulations of humic acid, fulvic acid, polysaccharides, and enzyme activity designed for Phytophthora blight control, thus failing to achieve the dual effects of efficient disease control and rapid plant recovery. 2) Mechanism gaps: Existing research focuses mainly on the soil-improving effects of humic acid, without fully revealing its synergistic mechanism with polysaccharides, cellulase, and other components in controlling Phytophthora blight, especially lacking research on multi-target mechanisms such as "stimulating systemic resistance in plants + interfering with pathogen cell walls + promoting wound healing". 3) Application gaps: Currently, there are no mature products on the market that clearly use humic acid as the core component to effectively control Phytophthora blight and achieve plant recovery.

[0005] Therefore, there is an urgent need to develop a biological control product with a well-defined formula, clear mechanism of action, stable control effect, and environmental friendliness to replace or supplement existing chemical control methods. This invention addresses the aforementioned technical deficiencies and gaps by providing a method for preparing a carbon chain enzymatic hydrolysate with specific components and processes. This method clarifies the content range of each component, establishes a standardized production process, and achieves highly efficient, stable, and environmentally friendly control of Phytophthora blight. Summary of the Invention

[0006] Purpose of the invention The primary objective of this invention is to provide a method for preparing a carbon chain enzymatic hydrolysate for the prevention and control of Phytophthora blight in plants. This method solves the problems of unclear formulations and unstable control effects of existing humic acid products by using precise raw material ratios and controllable enzymatic hydrolysis processes.

[0007] Another objective of this invention is to reveal the mechanism by which the carbon chain enzymatic hydrolysate, through the synergistic action of the components "polysaccharide-mineral-derived potassium humate-cellulase", stimulates the plant's own systemic resistance, interferes with pathogens, and promotes wound healing, providing a theoretical basis and technical guarantee for the biological control of Phytophthora blight in plants.

[0008] Another objective of this invention is to provide a biological control product that can replace traditional chemical fungicides, thereby reducing pesticide residues, delaying the development of pathogen resistance, reducing environmental pollution risks, and meeting the needs of green, ecological, and sustainable agricultural development.

[0009] Technical solution To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A carbon chain enzymatic hydrolysate for controlling Phytophthora blight in plants, the main components of which include water, polysaccharides, mineral-derived potassium humate, and cellulase. The components are prepared in the following weight ratio: water : polysaccharides : mineral-derived potassium humate : cellulase = 100 : 3-6 : 6-10 : 1-4.

[0010] The method for preparing the carbon chain enzymatic hydrolysate for controlling Phytophthora blight specifically includes the following steps: Step 1: Preparation of polysaccharide solution: Heat water to 100°C, then add polysaccharide at a ratio of 3-6:100, and stir until completely dissolved to form polysaccharide solution.

[0011] Step 2, mixing of potassium humate: Cool the polysaccharide solution obtained in Step 1 to 40°C, then add potassium humate of mineral source at a ratio of 6-10:100 to water, and stir continuously for 5 minutes to ensure thorough mixing.

[0012] Step 3, Enzymatic hydrolysis: Add cellulase to the mixture from Step 2 at a ratio of 1-4:100, keep the liquid temperature at no less than 30°C, and stir continuously to carry out the enzymatic hydrolysis reaction for 20 minutes.

[0013] Step 4: Static Aging and Finished Product Processing: After enzymatic hydrolysis, maintain the temperature of the mixture at no less than 30°C and allow it to stand for 12-20 hours for aging. After aging, dispense and package the product to obtain the carbon chain enzymatic hydrolysate.

[0014] Raw material requirements and efficacy • Water: It shall comply with the provisions of GB 5749-2022 "Standards for Drinking Water Quality".

[0015] • Polysaccharides: Their efficacy lies in their synergistic effect with fulvic acid and cellulase. The polysaccharides used should comply with the relevant provisions of Method I of GB 5009.7-2016 "Determination of Reducing Sugars in Food".

[0016] • Mineral-derived potassium humate: Its function is to promote the transport and translocation of nutrients within plants, thereby enhancing plant resistance to adverse conditions. The mineral-derived potassium humate used should comply with the requirements of GB / T 41732-2022 "Mineral-derived Potassium Humate".

[0017] • Cellulase: Its function is to efficiently degrade cellulose in plant diseased plant debris, accelerate cellulose mineralization, and eliminate the primary source of infection for pathogens. Simultaneously, through a synergistic effect, it indirectly participates in the lysis of the cell walls of pathogenic fungi. Food-grade cellulase with an enzyme activity ≥5000 U / g is preferred, derived from *Trichoderma reesei*, and meets the requirements of GB 28403-2012 "National Food Safety Standard for Food Additives: Cellulase Preparations".

[0018] Product quality control indicators • Physicochemical properties: ◦ Humic acid content ≥ 2000 mg / kg ◦ Fulvic acid content ≥ 800 mg / kg ◦ Polysaccharide content ≥ 100 mg / kg ◦ Cellulase activity ≥ 1.4 U / mL (Assay conditions: 37℃, pH 5.5, using sodium carboxymethyl cellulose as substrate, determined according to GB / T 23527-2009 method) ◦ Water-insoluble matter content ≤ 10 mg / L ◦ pH range: 3.0 - 9.0 • Sensory indicators: The product is a brownish-brown liquid, odorless, without layering, and without sediment.

[0019] • Microbiological indicators: ◦ Fecal coliform count ≤ 100 CFU / mL • Heavy metal limits: ◦ Cadmium (Cd) ≤ 3 mg / kg ◦ Mercury (Hg) ≤ 2 mg / kg ◦ Arsenic (As) ≤ 15 mg / kg ◦ Lead (Pb) ≤ 50 mg / kg ◦ Chromium (Cr) ≤ 150 mg / kg ◦ Thallium (Tl) ≤ 2.5 mg / kg Beneficial effects

[0020] Compared with the prior art, the present invention has the following outstanding advantages and beneficial effects: 1. Superior Recovery Capacity of Diseased Trees: Existing technologies mostly report the preventive or early control effects of humic acid on Phytophthora blight. However, this invention, through actual field application cases, demonstrates that the carbon chain enzymatic hydrolysate can enable severely infected durian trees with a trunk circumference exceeding 50% and a large number of leaves falling off the crown to sprout new shoots, form callus tissue, and restore tree vigor in a short period of time (e.g., 2 months), achieving a "significantly effective" recovery effect, overturning the traditional perception that "severely infected durian trees cannot recover."

[0021] 2. Superior Product Performance and Safety: Tests conducted by third-party testing institutions (such as Heilongjiang Huace Testing Technology Co., Ltd.) have shown that the actual effective components (such as humic acid, fulvic acid, polysaccharides, and cellulase activity) of the carbon chain enzymatic hydrolysate described in this invention significantly exceed the basic requirements of national and industry standards, providing a solid material basis for its excellent prevention and rehabilitation effects. Simultaneously, its microbial indicators (such as fecal coliform count) and heavy metal content (such as cadmium, chromium, mercury, lead, arsenic, and thallium) are far below or even undetectable by relevant standard limits, demonstrating that the product is highly effective while also possessing high environmental friendliness and biosafety.

[0022] 3. Unique Synergistic Mechanism: The effects of this invention are not a simple repetition of the known effects of humic acid. Its superior performance stems from the synergistic effect of a specific component system: polysaccharide-mineral-derived potassium fulvate-cellulase. Cellulase not only directly interferes with pathogens but also plays a crucial role in promoting the activation of wound healing signals in plants. This effect, combined with the systemic resistance induced by mineral-derived potassium fulvate and the pre-stimulation effect of polysaccharide on the plant immune system, produces a synergistic effect of "1+1+1 > 3," achieving outstanding multi-target (stimulating plant systemic resistance, interfering with pathogens, and promoting wound healing) prevention and rehabilitation effects.

[0023] 4. Environmental friendliness and sustainability: The product of this invention can effectively replace or reduce the use of chemical fungicides, thereby reducing pesticide residues, delaying the development of pathogen resistance, and reducing pollution to soil and water, which is in line with the development direction of green ecological agriculture. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0025] Example 1 Preparation of a carbon chain enzymatic hydrolysate for controlling Phytophthora blight in plants: • Raw material ratio: 100kg water, 4kg polysaccharide, 8kg mineral-derived potassium humate, 2kg cellulase.

[0026] • Preparation steps: 1. Heat 100kg of water to 100°C, add 4kg of polysaccharide, and stir until completely dissolved to obtain polysaccharide solution.

[0027] 2. Cool the above polysaccharide solution to 40°C, add 8 kg of mineral-derived potassium humate, and stir continuously for 5 minutes to mix it evenly.

[0028] 3. Add 2 kg of cellulase to the mixture, keep the liquid temperature above 30°C, and stir continuously to carry out the enzymatic hydrolysis reaction for 20 minutes.

[0029] 4. After enzymatic hydrolysis, maintain a temperature of no less than 30°C and let it stand for 16 hours to mature.

[0030] 5. After maturation, the product is divided and packaged to obtain the carbon chain enzymatic hydrolysate.

[0031] Example 2 Preparation of a carbon chain enzymatic hydrolysate for controlling Phytophthora blight in plants: • Raw material ratio: 100kg water, 3kg polysaccharide, 10kg mineral-derived potassium humate, 1kg cellulase.

[0032] • Preparation steps: Refer to Example 1, wherein 3 kg of polysaccharide is added in step one, 10 kg of mineral-derived potassium humate is added in step two, and 1 kg of cellulase is added in step three, and the mixture is allowed to stand for 12 hours to mature.

[0033] Example 3 Preparation of a carbon chain enzymatic hydrolysate for controlling Phytophthora blight in plants: • Raw material ratio: 100kg water, 6kg polysaccharide, 6kg mineral-derived potassium humate, 4kg cellulase.

[0034] • Preparation steps: Refer to Example 1, wherein 6 kg of polysaccharide is added in step one, 6 kg of mineral-derived potassium humate is added in step two, and 4 kg of cellulase is added in step three, and the mixture is allowed to stand and mature for 20 hours.

[0035] Application Examples The carbon chain enzymatic hydrolysate prepared in Examples 1-3 above can be applied as a foliar spray, root drenching, or trunk smear at the recommended dilution ratio (e.g., 500-1000 times) to control durian canker, citrus brown rot, and other diseases. Field trials have shown that this product can effectively control disease development, promote wound healing and new tissue growth in diseased plants, and significantly restore tree vigor.

[0036] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a carbon chain enzymatic hydrolysate for controlling Phytophthora blight in plants, characterized in that, It is prepared from water, polysaccharides, mineral-derived potassium humate, and cellulase as essential raw materials.

2. The method for preparing a carbon chain enzymatic hydrolysate for controlling Phytophthora blight according to claim 1, characterized in that, The weight ratio of water, polysaccharide, mineral-derived potassium humate, and cellulase is 100 : 3-6 : 6-10 : 1-4.

3. The method for preparing a carbon chain enzymatic hydrolysate for controlling Phytophthora blight according to claim 1 or 2, characterized in that, Includes the following steps: Step 1: Heat water to 100°C, then add polysaccharides according to the formula, stir to dissolve, and form a polysaccharide solution; Step 2: Cool the polysaccharide solution from Step 1 to 40°C, then add potassium humate (mineral source) according to the ratio, and stir for 5 minutes to mix it evenly. Step 3: Add cellulase to the mixture from Step 2 according to the specified ratio, keep the liquid temperature at no less than 30°C, and stir continuously for 20 minutes for enzymatic hydrolysis. Step 4: After enzymatic hydrolysis, keep the liquid temperature at no less than 30°C and let it stand for 12-20 hours to mature, then dispense and package.

4. The method for preparing a carbon chain enzymatic hydrolysate for controlling Phytophthora blight according to claim 1 or 2, characterized in that, The polysaccharide conforms to the requirements of Method I of GB 5009.7-2016 "Determination of reducing sugars in food".

5. The method for preparing a carbon chain enzymatic hydrolysate for controlling Phytophthora blight according to claim 1 or 2, characterized in that, The mineral-derived potassium humate conforms to the requirements of GB / T 41732-2022 "Mineral-derived Potassium Humate".

6. A method for preparing a carbon chain enzymatic hydrolysate for controlling Phytophthora blight according to claim 1 or 2, characterized in that, The cellulase is food grade, with an enzyme activity ≥5000 U / g, and is derived from Trichoderma reesei, meeting the requirements of GB 28403-2012 "National Food Safety Standard for Food Additives: Cellulase Preparations".

7. A method for preparing a carbon chain enzymatic hydrolysate for controlling Phytophthora blight according to claim 1 or 2, characterized in that, The water meets the requirements of GB 5749-2022 "Standards for Drinking Water Quality".

8. A method for preparing a carbon chain enzymatic hydrolysate for controlling Phytophthora blight according to claim 1 or 2, characterized in that, Its quality meets the following indicators: Humic acid content ≥ 2000 mg / kg; Fulvic acid content ≥ 800 mg / kg; Polysaccharide content ≥ 100 mg / kg; Cellulase activity ≥ 1.4 U / mL (measured at 37℃ and pH 5.5); Water-insoluble matter content ≤ 10 mg / L; The pH range is 3.0-9.0; Fecal coliform count ≤ 100 CFU / mL; Heavy metal content: cadmium ≤ 3 mg / kg, mercury ≤ 2 mg / kg, arsenic ≤ 15 mg / kg, lead ≤ 50 mg / kg, chromium ≤ 150 mg / kg, thallium ≤ 2.5 mg / kg.

9. The method for preparing the carbon chain enzymatic hydrolysate for controlling Phytophthora blight as described in any one of claims 1 to 8 is used in the preparation of formulations for controlling plant diseases caused by Phytophthora pathogens.

10. The application according to claim 9, characterized in that, The plant diseases mentioned are durian canker, citrus brown rot, or potato late blight. A carbon chain enzymatic hydrolysate for controlling Phytophthora blight in plants, characterized in that: The main components are water, polysaccharides, mineral-derived potassium humate, and cellulase.