Nutrition regulation and control method for disease resistance of kiwi fruits, germination fertilizer and preparation method

Abstract

The invention discloses a nutrition regulation and control method for disease resistance of kiwi fruits, a germination fertilizer and a preparation method, and the preparation method comprises the following steps: constructing a ternary interaction system consisting of a sulfur source, succinic acid and Pseudomonas fluorescens, and applying the ternary interaction system to rhizosphere nutrition management of the kiwi fruits in early spring. And three-dimensional coupling of nutrition supply, signal activation and microbial regulation and control is synchronously realized in the rhizosphere microdomain. Meanwhile, lignin monomer synthesis is continuously activated and promoted through a salicylic acid signal channel, high-activity laccase serves as a key enzyme for lignin monomer polymerization, lignin monomer polymerization reaction is accelerated, synthesis and deposition of lignin in plant cell walls are promoted, plant endogenous disease-resistant signal cascade is continuously amplified, and plant disease resistance is improved. The kiwi fruits are induced to generate stable systematic disease resistance. The composition can prevent and control diseases for a long time and cooperatively promote plant growth.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a method for regulating nutritional resistance to bacterial canker in kiwifruit, the germination fertilizer used, and its preparation method. Background Technology

[0002] Kiwifruit bacterial canker (KBC) is a devastating disease caused by Pseudomonas syringae pv. actinidiae (Psa). It mainly infects the stems of kiwifruit plants, causing them to decline in growth, die, and suffer significant yield reductions. In severe cases, it can even lead to the destruction of entire orchards, posing a serious threat to the sustainable development of the kiwifruit industry.

[0003] Currently, the control methods for this disease still mainly rely on copper-based and antibiotic pesticides. However, long-term continuous use of these chemical agents can easily lead to the pathogens gradually developing drug resistance, resulting in a year-by-year decline in control effectiveness. At the same time, it can also cause pesticide residues to exceed the standards, polluting the soil, water sources and other ecological environments, which is contrary to the concept of green agricultural development and makes it difficult to meet the needs of high-quality industrial development.

[0004] Traditional kiwifruit sprouting fertilizers have a relatively singular function, focusing only on nutrient supply. They fail to effectively integrate the three core functions of nutrient supply, rhizosphere microecological regulation, and immune induction, making it difficult to achieve synergistic improvement in disease control and plant growth and development, and thus unable to fundamentally solve the problem of bacterial canker control. Summary of the Invention

[0005] The main objective of this invention is to address bacterial canker in kiwifruit caused by pathogenic species of *Pseudomonas syringae*. A ternary interaction system, constructed from a sulfur source, succinic acid, and a specific fluorescent *Pseudomonas* bacterium, is applied to early spring rhizosphere nutrient management in kiwifruit. This system achieves three-dimensional coupling of nutrient supply, signal activation, and microbial regulation, thereby activating the systemic disease resistance of kiwifruit, providing long-term disease control, and synergistically promoting plant growth.

[0006] Based on the first major aspect of the present invention, a nutritional regulation method for activating systemic disease resistance in kiwifruit is provided. The method targets bacterial canker disease in kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa). The method includes constructing a ternary interaction system composed of a sulfur source, succinic acid, and Pseudomonas fluorescens, and applying the ternary interaction system to early spring rhizosphere nutrient management of kiwifruit, thereby simultaneously achieving three-dimensional coupling of nutrient supply, signal activation, and microbial regulation in the rhizosphere microdomain.

[0007] The sulfur source is used to initiate systemic disease resistance pathways in kiwifruit and activate the salicylic acid signaling pathway; the succinic acid is used to enhance the chemotaxis and biofilm formation ability of Pseudomonas fluorescens to promote its colonization in the rhizosphere, and further amplify the salicylic acid signaling pathway cascade reaction through the colonized Pseudomonas fluorescens; the sulfur source, succinic acid and Pseudomonas fluorescens synergistically upregulate the expression level of laccase gene in kiwifruit and enhance laccase activity.

[0008] Meanwhile, the salicylic acid signaling pathway is continuously activated to promote lignin monomer synthesis. Highly active laccase, as a key enzyme in lignin monomer polymerization, accelerates the lignin monomer polymerization reaction, promotes the synthesis and deposition of lignin in the plant cell wall, continuously amplifies the endogenous disease resistance signal cascade in the plant, and induces stable systemic disease resistance in kiwifruit.

[0009] The *Pseudomonas fluorescens* used in this invention is an environmental pollutant. *Pseudomonas fluorescens* belongs to the genus *Pseudomonas*, is a chemoheterotrophic Gram-negative rod-shaped bacterium with flagella. It secretes a yellow-green fluorescent pigment to emit fluorescence and produces metabolic products such as antibiotics and hydrolases. It grows in a neutral environment within the temperature range of 4℃-37℃ and exhibits significant physiological and biochemical characteristics. The role of *Pseudomonas fluorescens* in this invention is similar to the principle of vaccination. It can amplify the cascade reaction of the salicylic acid signaling pathway activated by sulfur sources, synergistically upregulate the expression of the laccase gene and increase laccase activity in kiwifruit with sulfur sources and succinic acid, thereby promoting lignin synthesis and deposition, ultimately inducing stable systemic disease resistance in kiwifruit and controlling bacterial canker.

[0010] From a holistic perspective, this invention targets ulcer disease caused by pathogenic species of *Pseudomonas syringae* in kiwifruit. It constructs a ternary interaction system consisting of a sulfur source, succinic acid, and *Pseudomonas fluorescens*, and applies it to early spring rhizosphere nutrient management of kiwifruit, achieving three-dimensional coupling of nutrient supply, signal activation, and microbial regulation.

[0011] Simultaneously, by activating systemic disease resistance-related pathways and salicylic acid (SA) signaling pathways through sulfur sources, succinic acid enhances the chemotaxis and biofilm formation ability of Pseudomonas fluorescens to promote rhizosphere colonization. After colonization, the strain further amplifies the SA signaling cascade reaction. The three synergistically upregulate laccase gene expression and enhance laccase activity. At the same time, the continuous activation of the SA signaling pathway promotes lignin monomer synthesis, and highly active laccase accelerates monomer polymerization, promoting lignin deposition in the cell wall.

[0012] Therefore, with the overall synergy of the above schemes, the cascade of endogenous disease resistance signals in plants can be continuously amplified, inducing kiwifruit to produce stable systemic disease resistance and effectively controlling bacterial canker.

[0013] In some embodiments, preferably, the sulfur source is elemental sulfur, the succinic acid has a purity ≥98%, and is a food-grade or agricultural-grade product; the fluorescent Pseudomonas includes similar strains isolated from the rhizosphere soil of sulfur-treated kiwifruit, possessing strong chemotaxis and high biofilm formation ability, and the effective viable count of the functional bacterial solution prepared therefrom is ≥1×10⁻⁶. 8 CFU / mL.

[0014] In the above scheme, by standardizing the morphology, purity, strain characteristics and bacterial concentration of key components, it is ensured that each component is adapted to the synergistic logic of the ternary interaction system, thereby avoiding problems such as low strain colonization efficiency and weak SA signal activation caused by non-compliant components, ensuring the stability and efficiency of the ternary interaction system, and providing a basic guarantee for the disease resistance effect.

[0015] In some embodiments, preferably, applying the ternary interaction system to the early spring rhizosphere nutrient management of kiwifruit includes preparing a special kiwifruit sprouting fertilizer using sulfur source, succinic acid and Pseudomonas fluorescens as part of the raw materials and applying it to kiwifruit.

[0016] In the above scheme, the abstract nutrient regulation method is transformed into a practical fertilizer form that is suitable for the early spring fertilization scenario of kiwifruit. The three-dimensional coupling of nutrient supply and disease resistance regulation is achieved through conventional fertilization operations, thereby simplifying the technology application process, lowering the threshold for farmers to use, and allowing nutrient supplementation during the kiwifruit budding period and bacterial canker disease control to be carried out simultaneously, thereby improving the field operability and practicality of the technology.

[0017] More preferably, the ternary interaction system adopts a stepwise application sequence optimization: first, the sulfur source is mixed with well-rotted organic fertilizer and applied to the rhizosphere of kiwifruit for 7-10 days to improve the rhizosphere microenvironment and initiate the initial salicylic acid signal; then, succinic acid is mixed with Pseudomonas fluorescens bacterial solution and applied, so that succinic acid first provides suitable conditions for the colonization of the strain. After the strain colonizes, it synergistically amplifies the salicylic acid signaling pathway with the continuously released sulfur source, further improving the laccase gene expression efficiency and lignin deposition uniformity.

[0018] The above scheme first creates suitable conditions for the colonization of the strain with succinic acid. After colonization, the strain synergistically amplifies the SA signaling pathway with the continuously released sulfur source. This meets the progressive requirements of improving the rhizosphere microenvironment, colonizing the strain, and activating the signal. It can significantly improve the expression efficiency of laccase genes and the uniformity of lignin deposition, avoid the weakening of synergistic effect due to improper application timing, ensure the continuous and stable activation of disease resistance signals, and enhance the stability of the control effect.

[0019] More preferably, the sulfur source is modified by coating, and the coating material is a biodegradable starch-chitosan composite membrane with a coating thickness controlled at 50-100 μm. Through coating, the sulfur source is slowly released in the rhizosphere microdomain within 12 months, and the release rate matches the colonization cycle of Pseudomonas fluorescens and the activation rhythm of the salicylic acid signaling pathway.

[0020] The above scheme can effectively prevent the decline of disease resistance due to premature depletion of sulfur source, prolong the duration of action of ternary interaction system, and ensure that the ulcer disease prevention and control effect is maintained stably for at least 12 months.

[0021] More preferably, the fluorescent Pseudomonas bacterial suspension is pretreated with a low concentration of salicylic acid before use: the bacterial suspension is mixed with salicylic acid at a concentration of 10-20 ng / mL and incubated at 25-30°C for 6-8 hours.

[0022] The above schemes enhance the chemotactic response of the strain to succinic acid and the rate of biofilm formation through pretreatment, accelerate the colonization of Pseudomonas fluorescens in the rhizosphere of kiwifruit, improve its efficiency in activating the SA signaling pathway, shorten the initiation cycle of lignin synthesis and deposition, and allow the disease resistance effect to appear earlier.

[0023] Based on the second main aspect of the present invention, a special budding fertilizer for kiwifruit that controls bacterial canker is provided, the raw materials of which include a ternary interaction system composed of a sulfur source, succinic acid and Pseudomonas fluorescens.

[0024] A ternary interaction system composed of sulfur source, succinic acid, and *Pseudomonas fluorescens* is incorporated into the raw materials of a special kiwifruit sprouting fertilizer. This allows the fertilizer to simultaneously provide nutrition and control bacterial canker, overcoming the limitation of traditional sprouting fertilizers that only focus on nutrition. This system can synergistically promote nutrient supplementation and bacterial canker control during the kiwifruit sprouting period, eliminating the need for additional pesticides or disease-resistant agents, reducing planting costs, and aligning with the concept of green agriculture.

[0025] In some embodiments, preferably, the raw material composition of the sprouting fertilizer, calculated by mass percentage, is: 18-25 parts of 46% urea, 6-10 parts of 57% diammonium phosphate, 9-13 parts of granulated potassium sulfate, 3-7 parts of elemental sulfur, 10-20 parts of organic fertilizer, 0.5-2 parts of functional bacterial solution, and 0.5-2 parts of succinic acid; wherein the functional bacterial solution is prepared from one or more fluorescent Pseudomonas bacteria.

[0026] The above ratio not only meets the nutritional needs of kiwifruit during the budding stage for nitrogen, phosphorus, potassium, etc., promoting plant growth, but also fully activates the disease resistance mechanism through the precisely proportioned ternary system, achieving the dual goals of growth and disease prevention.

[0027] Based on the third main aspect of the present invention, a method for preparing the aforementioned kiwifruit sprouting fertilizer for preventing and controlling bacterial canker is provided, comprising the following steps:

[0028] (1) The organic fertilizer is decomposed for 10–12 days, and the moisture content of the organic fertilizer after treatment is controlled to be 45–55% and the pH value is 6.0–6.8;

[0029] (2) Add functional bacterial solution and succinic acid to the organic fertilizer treated in step (1), stir evenly and let stand for 24 hours to promote bacterial activation;

[0030] (3) Mix 46% urea, 57% diammonium phosphate, granulated potassium sulfate and elemental sulfur, crush and sieve, with a sieve hole size ≤3mm, then dry to a moisture content ≤12% and complete packaging.

[0031] In the above scheme, the organic fertilizer is first composted to a specific moisture content (45-55%) and pH (6.0-6.8). Then, functional bacterial solution and succinic acid are added to activate the bacteria. Finally, nitrogen, phosphorus, and potassium fertilizers are mixed with elemental sulfur, crushed, sieved (≤3mm), dried (moisture content ≤12%), and packaged. The parameters of each step are adapted to the characteristics of the components, thereby ensuring that the organic fertilizer is fully composted, the functional bacteria are sufficiently active, the components are evenly mixed, and nutrients are not lost, thus ensuring the stable and consistent nutrient supply capacity and disease resistance regulation effect of the germination fertilizer.

[0032] In some embodiments, preferably, the composting of organic fertilizer in step (1) is carried out by composting fermentation, with the fermentation environment temperature controlled at 25-35℃, and the compost is turned over every 2-3 days; the drying temperature in step (3) is 50-60℃, and a low-temperature hot air drying method is used to avoid the volatilization of elemental sulfur and loss of nutrients caused by high temperature.

[0033] The above solutions specifically address the problems of uneven composting, volatilization of elemental sulfur caused by high temperatures, and nutrient loss. They can improve the quality of organic fertilizer composting, ensure that functional components and nutrients are not destroyed during the preparation process, further optimize the quality stability of sprouting fertilizer, and guarantee its nutrient supply efficiency and disease control effect when applied in the field.

[0034] Compared with existing technologies, this invention first breaks through the traditional reliance on copper-based and antibiotic pesticides for the control of bacterial canker in kiwifruit. Through the construction of a ternary interaction system of sulfur source-succinic acid-fluorescent Pseudomonas aeruginosa, it achieves a green control model that replaces pesticides with fertilizers. Compared with existing chemical control methods, this system activates the plant's own systemic disease resistance from the rhizosphere microdomain, avoiding the problems of increased pathogen resistance, excessive pesticide residues, and environmental pollution caused by long-term pesticide use. It aligns with the concept of green agriculture development and provides a new path for the ecological control of fruit tree diseases.

[0035] Secondly, this invention addresses the core deficiency of existing kiwifruit sprouting fertilizers, which have a single function. For the first time, it achieves the synergistic coupling of three major functions: nutrient supply, rhizosphere microecological regulation, and immune induction. Existing fertilizers only focus on supplementing nutrients such as nitrogen, phosphorus, and potassium. However, this invention, through a ternary interaction system, continuously activates the salicylic acid signaling pathway, upregulates laccase gene expression, and promotes lignin synthesis and deposition. While meeting the nutritional needs of kiwifruit during sprouting and promoting plant growth, it also forms a stable physical barrier and molecular regulatory mechanism against disease, ensuring that the control effect against citrus canker is stably maintained for at least 12 months, breaking through the bottleneck of traditional technologies that are unstable in their prevention.

[0036] Finally, this invention further enhances the practicality and stability of its effects by optimizing application timing, modifying sulfur source coating, and pretreating bacterial solutions. Compared to existing fertilization technologies that lack precise control, this invention transforms the ternary interaction system into a dedicated germination fertilizer form. Combined with step-by-step application, this lowers the application threshold while ensuring the rhythmic matching of sulfur source release, bacterial colonization, and signal activation. This results in a 17%-52% reduction in ulcerative disease incidence compared to conventional fertilization groups, an increase of ≥40% in leaf salicylic acid content, and an increase of ≥2.6 times in laccase activity. This achieves a dual improvement in disease control and plant growth, significantly outperforming the single-function effects of existing technologies. Detailed Implementation

[0037] The preferred embodiments of the present invention will be described in detail below to provide a clearer understanding of the purpose, features, and advantages of the invention. It should be understood that the following embodiments are not intended to limit the scope of the invention, but are merely illustrative of the essential spirit of the technical solution of the invention.

[0038] In the following description, certain specific details are set forth for the purpose of illustrating various disclosed embodiments in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the art will recognize that embodiments may be practiced without one or more of these specific details. In other instances, well-known techniques associated with the invention may not have been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0039] Throughout this specification, references to "an embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Therefore, the appearance of "in an embodiment" or "an embodiment" in various places throughout the specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any manner in one or more embodiments.

[0040] As a basic approach, this embodiment was applied to a kiwifruit plantation. Targeting the bacterial canker disease caused by *Pseudomonas syringae* pv. actinidiae (Psa) infection, which had plagued the plantation for many years, five-year-old *Actinidia chinensis* var. *syringae* were selected as the experimental subjects. The experimental area was 10 mu (approximately 1.65 acres), with three replicates. A control group was also set up with conventional fertilization (without the ternary interaction system), while all other field management conditions remained consistent.

[0041] The specific selection of each component in the ternary interaction system is as follows: the sulfur source is elemental sulfur with a particle size ≤3mm and a purity ≥99%; succinic acid is an agricultural grade product with a purity of 98.5%; the fluorescent Pseudomonas is Pseudomonas fluorescens MK7 isolated from the rhizosphere soil of sulfur-treated kiwifruit. This strain is an existing fluorescent Pseudomonas, and MK7 is the identification number determined after the collection of this strain. In actual implementation, similar fluorescent Pseudomonas collected under similar conditions can be used to achieve the purpose of this embodiment. Functional bacterial solutions were prepared through laboratory-scale culture, and the effective viable count was detected to be 1.2 × 10⁻⁶. 8 CFU / mL ensures that the strain has strong chemotaxis and high biofilm formation ability.

[0042] In this embodiment, the ternary interaction system was constructed as follows: First, elemental sulfur was mixed evenly with an appropriate amount of well-rotted organic fertilizer (rotation degree ≥90%) as a basic nutrient-signal initiation substrate. Then, succinic acid and *Pseudomonas fluorescens* MK7 bacterial solution were mixed at a mass ratio of 1:1, stirred evenly, and allowed to stand for 6 hours to allow the succinic acid and bacterial strain to fully adapt, enhancing the strain's responsiveness to the rhizosphere environment. Finally, the two mixtures were integrated to form a complete sulfur source-succinic acid-*Pseudomonas fluorescens* ternary interaction system, ensuring that each component can quickly form a synergistic effect in the rhizosphere microdomain after application.

[0043] The application time is 12 days before the kiwifruit buds sprout in early spring. Nutrient regulation is carried out by rhizosphere trench application. A circular trench with a depth of 20cm and a width of 15cm is dug inside the drip line of the canopy of each kiwifruit tree. The constructed ternary interaction system is evenly spread in the trench, with an application rate of 3kg per tree. Then, the surface soil is covered and the roots are thoroughly watered to ensure that the system components are in full contact with the rhizosphere soil. This creates a suitable rhizosphere microenvironment for sulfur source release, bacterial colonization and signal activation, and simultaneously initiates the three-dimensional coupling process of nutrient supply (sulfur source and organic fertilizer provide basic nutrition), signal activation (sulfur source triggers initial salicylic acid signal) and microbial regulation (succinic acid assists bacterial colonization).

[0044] Three months after application, samples were taken from the kiwifruit plants in the experimental group for testing. The results showed that the endogenous salicylic acid content in the roots, stems, and leaves reached 26.3 ng / g, 21.8 ng / g, and 95.2 ng / g, respectively, which were 43.7%, 43.2%, and 31.3% higher than the control group. This confirmed that the sulfur source successfully activated the salicylic acid signaling pathway, and that succinic acid promoted the efficient colonization of *Pseudomonas fluorescens* MK7 in the rhizosphere (colonization density reached 1.5 × 10⁻⁶). 6 The CFU / g soil concentration further amplified the signal cascade reaction. Simultaneously, the abundance of beneficial microbial communities in the rhizosphere microdomain of the plants was significantly increased, and the detection rate of harmful pathogen Psa decreased by 41% compared to the control group, demonstrating the significant effect of microbial regulation.

[0045] Nine months after application, the laccase activity and lignin synthesis in the kiwifruit plants of the experimental group were measured. The results showed that laccase activity increased from the initial 1180 U / g to 1550 U / g, a 31.4% increase compared to the control group, indicating that sulfur source, succinic acid, and *Pseudomonas fluorescens* MK7 synergistically upregulated the expression level of the laccase gene. The lignin monomer content reached 129.5 μg / mL, a 27.4% increase compared to the control group. Furthermore, observation of sections revealed that lignin formed a uniform deposition in the cell walls of the plant stems, with the cell wall thickness increasing by 23% compared to the control group, effectively constructing a physical disease resistance barrier and continuously amplifying the cascade of endogenous disease resistance signals in the plant.

[0046] Twelve months after application, the incidence of bacterial canker and plant growth in the experimental and control groups were statistically analyzed: the incidence of bacterial canker in the experimental group of kiwifruit was 20.5%, which was 51.3% lower than that in the control group (71.8%), and the lesion area of ​​the diseased plants was significantly reduced, with no whole plant dying.

[0047] Meanwhile, the germination rate of kiwifruit in the experimental group reached 92%, which was 15% higher than that in the control group. The average length of new shoots increased by 22cm, and the chlorophyll content of leaves increased by 18%. This confirms that the nutrient regulation method can effectively promote plant growth and development through the synergistic effect of nutrient supply and disease resistance regulation, while inducing stable systemic disease resistance in kiwifruit, thus achieving the dual goals of disease control and growth improvement.

[0048] As a further preferred embodiment, in the following possible embodiments, the sulfur source is elemental sulfur with a particle size ≤3mm and a purity ≥99%, ensuring the effective release and utilization of sulfur. The succinic acid is selected as an agricultural-grade product with a purity of 98.8%, free of harmful impurities, and suitable for agricultural production needs. The *Pseudomonas fluorescens* strain is selected from the rhizosphere soil of sulfur-treated kiwifruit. Laboratory tests show that this strain has a higher chemotactic value than ordinary *Pseudomonas fluorescens*, and a significantly increased biofilm formation, meeting the characteristics of strong chemotaxis and high biofilm formation ability.

[0049] Functional bacterial culture was prepared by liquid fermentation and scale-up culture. The effective viable count was stable at 1.5 × 10⁻⁶ using the plate count method. 8 CFU / mL, satisfying ≥1×10 8 Limits on CFU / mL.

[0050] The above components were constructed as a ternary interaction system and applied via rhizosphere hole application. By clarifying the sulfur source morphology, succinic acid purity level, and the source and core characteristics of *Pseudomonas fluorescens*, and quantifying the effective viable count in the bacterial solution, the problem of weakened synergistic effects caused by ambiguous component parameters was avoided.

[0051] Compared to the basic scheme, the colonization density of *Pseudomonas fluorescens* in the rhizosphere of kiwifruit was increased to 2.1 × 10⁻⁶ in this embodiment. 6 The CFU / g soil concentration was increased by 40% compared to the basic treatment. The activation efficiency of the salicylic acid signaling pathway was enhanced, and the endogenous salicylic acid content in leaves was increased by an additional 12% compared to the basic treatment. The incidence of ulcerative canker was further reduced by 8% compared to the basic treatment, effectively ensuring the stability and reliability of the ternary interaction system and providing precise component-level protection for disease control.

[0052] As a further preferred embodiment, in the following possible embodiments, each component of the ternary interaction system is used as the core functional raw material, and is combined with 22 parts of 46% urea, 8 parts of 57% diammonium phosphate, 11 parts of granulated potassium sulfate, and 15 parts of organic fertilizer to prepare a special kiwifruit sprouting fertilizer according to the mass percentage ratio.

[0053] In the preparation process, elemental sulfur (5 parts) is first mixed with well-rotted organic fertilizer, and then succinic acid (1 part) and Pseudomonas fluorescens MK7 bacterial solution (1 part, effective viable count 1.2 × 10⁻⁶) are added. 8 After stirring evenly, let stand for 24 hours to activate the bacteria. Finally, mix with nitrogen, phosphorus and potassium fertilizer, crush and sieve (≤3mm), dry at low temperature until the moisture content is ≤10% and then package.

[0054] When applying, apply 3 kg per plant in a root trench 10 days before the kiwifruit sprouts, and then cover with soil and water.

[0055] In this embodiment, the abstract ternary interaction system is transformed into a standardized, specialized sprouting fertilizer, eliminating the need for growers to individually mix each component, simplifying the operation process and lowering the technical application threshold. Compared to the basic scheme of applying each component separately, the sprouting fertilizer of this embodiment has a more uniform distribution of each component in the rhizosphere microdomain, increasing the synergistic response speed of nutrient supply and disease resistance regulation by 20%, improving the nutrient absorption efficiency of kiwifruit during the sprouting period by 15%, and improving the uniformity of new shoot sprouting by 25%. At the same time, the control effect of canker disease is on par with the basic scheme, achieving the integrated goal of fertilization and disease prevention, which is more in line with the actual production needs of large-scale kiwifruit cultivation.

[0056] As a further preferred embodiment, a step-by-step application timing optimization strategy is employed in the following possible embodiments:

[0057] The first step is to mix elemental sulfur with well-rotted organic fertilizer at a mass ratio of 1:3 20 days before the kiwifruit sprouts. Dig a circular trench (25cm deep and 20cm wide) along the drip line of the tree canopy and apply the mixture. Apply 2kg per tree. After covering with soil and watering, pre-treat for 8 days to improve the root microenvironment.

[0058] The second step involves mixing succinic acid with *Pseudomonas fluorescens* bacterial suspension (effective viable count 1.3 × 10⁻⁶) 12 days before germination. 8 Mix (CFU / mL) at a mass ratio of 1:1.2, let stand for 4 hours, then apply as a supplementary treatment along the same circular trench, using 1 kg per plant, and cover with soil again and water thoroughly. The selection of other components and field management are the same as the basic plan.

[0059] In this embodiment, by optimizing the timing of pretreatment followed by supplementary application, the sulfur source was able to initiate the initial salicylic acid signal in advance, creating a suitable rhizosphere environment for bacterial colonization. After supplementary application of succinic acid and bacterial solution, the colonization rate of the strain increased by 30% compared to the basic scheme, and the colonization stability was enhanced, avoiding the rhizosphere microenvironment discomfort problem that may occur when all components are applied simultaneously. Compared to the basic scheme, the laccase gene expression efficiency was increased by 25% in this embodiment, and the laccase activity was increased by an additional 320 U / g. The uniformity of lignin deposition in the stem cell wall was improved by 40%, with no local insufficient deposition. The duration of the disease resistance signal activation was extended to 14 months, and the incidence of canker at 12 months was reduced by 6% compared to the basic scheme, significantly enhancing the stability and durability of the control effect.

[0060] As a further preferred embodiment, in the following possible embodiments, the sulfur source is coated and modified. Starch and chitosan are mixed at a mass ratio of 7:3, and an appropriate amount of deionized water is added to prepare a composite membrane solution. Elemental sulfur particles (particle size 2-3 mm) are immersed in the membrane solution and spray-dried to form a coating layer with a coating thickness controlled at 80 μm. Release tests show that the coated sulfur source has a release cycle of 12 months in the rhizosphere microdomain. The release amount accounts for 30% of the total content in the first 3 months, 45% from April to September, and 25% from October to December. The release rate precisely matches the colonization cycle of *Pseudomonas fluorescens* (rapid colonization in the first 3 months, stable colonization from April to September, and maintenance colonization from October to December) and the activation rhythm of the salicylic acid signaling pathway.

[0061] In this embodiment, the coating modification treatment avoids the problem of excessively high concentration in the early stage and insufficient supply in the later stage caused by the rapid release of elemental sulfur, and realizes the slow and continuous release of sulfur source. Compared with the uncoated sulfur source in the basic scheme, the effective utilization rate of sulfur in the rhizosphere is increased by 35% in this embodiment, and the colonization period of Pseudomonas fluorescens is extended by 3 months, still maintaining 1.2×10 at 12 months. 6 The colonization density of CFU / g soil was increased by 50% compared to the basic scheme. The salicylic acid signaling pathway remained in a highly activated state. At 12 months, the endogenous salicylic acid content in leaves was increased by an additional 18% compared to the basic scheme. Lignin synthesis continued to increase. At 12 months, the lignin monomer content was increased by 22% compared to the basic scheme. The control effect of canker disease did not decrease.

[0062] As a further preferred embodiment, in the following possible embodiments, the *Pseudomonas fluorescens* bacterial suspension was pretreated with a low concentration of salicylic acid before use. A salicylic acid solution with a concentration of 15 ng / mL was mixed with the *Pseudomonas fluorescens* MK7 bacterial suspension at a volume ratio of 1:10 and incubated in a 28°C incubator for 7 hours, with stirring every 1.5 hours to ensure sufficient contact between the bacterial suspension and the salicylic acid. The pretreated bacterial suspension, after testing, showed a 38% increase in chemotactic response and a 30% acceleration in biofilm formation rate compared to the untreated suspension. This pretreated bacterial suspension was then mixed with succinic acid to construct a ternary interaction system and applied.

[0063] In this embodiment, pretreatment with low-concentration salicylic acid activated the functional characteristics of *Pseudomonas fluorescens*, enhancing its chemotactic response to succinic acid and its biofilm formation ability. Compared to the basic method, the colonization initiation time of *Pseudomonas fluorescens* in the rhizosphere was shortened to 3 days in this embodiment, 4 days earlier than the basic method. The colonization density reached 1.8 × 10⁻⁶ m² / h⁻¹ one month after application. 6 CFU / g soil, the activation cycle of the salicylic acid signaling pathway is advanced by 2 weeks, the early disease resistance of plants is formed rapidly, the incidence of canker disease is reduced by 10% at 3 months compared with the basic program, the initiation cycle of lignin synthesis and deposition is shortened by 1 month, the physical barrier of cell wall is completed earlier, effectively resisting the initial infection of pathogens in early spring, and significantly improving the timeliness of disease control.

[0064] The following is a preferred embodiment, which aims to solve the problems of single fertilizer function, reliance on pesticides for disease control, and lack of rhizosphere microecological regulation in the prior art by using an organic-inorganic compound sprouting fertilizer with the function of preventing and controlling bacterial canker in kiwifruit, and by activating the systemic disease resistance of plants through sulfur-induced rhizosphere effects.

[0065] To achieve the above objectives, this embodiment provides the following germination fertilizer formula:

[0066] 1. Fertilizer formula (by parts by weight):

[0067] 46% urea: 24 parts;

[0068] 57% diammonium phosphate: 7 parts;

[0069] Granular potassium sulfate: 10 parts;

[0070] Elemental sulfur (S): 5 parts;

[0071] Organic fertilizer: 15 parts;

[0072] Functional bacterial solution: 1.5 parts;

[0073] Succinic acid: 1.5 parts;

[0074] The remainder consists of filler material, 30 parts in total. The filler material used in this invention can be either humic acid or diatomaceous earth; in this embodiment, humic acid is used.

[0075] 2. Sources of functional bacteria

[0076] The functional bacterium was Pseudomonas fluorescens MK7, isolated from the rhizosphere soil of sulfur-treated kiwifruit. It exhibited strong chemotaxis, high biofilm formation ability, and the ability to induce SA signaling pathway activation.

[0077] 3. Preparation method

[0078] (1). Organic fertilizer is composted for 10–12 days, with moisture content controlled at 45–55% and pH at 6.0–6.8;

[0079] (2). Add functional bacterial solution and succinic acid, stir well, and let stand for 24 hours to promote bacterial activation;

[0080] (3) Mix nitrogen, phosphorus and potassium fertilizers and elemental sulfur, crush and sieve (≤3mm), dry to a moisture content of ≤12%, add filler and package.

[0081] The implementation effect of this embodiment is as follows:

[0082] 1. Significantly reduced incidence of kiwifruit canker. After applying the formula of this invention for 3, 6, 9, and 12 months, the incidence of kiwifruit canker was 20%–28%, which was 17%–52% lower than the control group treated with conventional fertilization. This demonstrates that the formula of this embodiment can continuously reduce the incidence of kiwifruit canker, confirming the inhibitory effect of the sulfur-succinic acid-functional bacteria ternary system on natural infection in the field.

[0083] Table 1: Comparison of the incidence rate of peptic ulcer disease between this embodiment and the control example.

[0084]

[0085] 2. Rapid amplification of salicylic acid signal. After applying the formula of this invention, the salicylic acid content of the entire kiwifruit plant increased, and data after 3, 6, 9, and 12 months showed that it continued to increase. In particular, the endogenous salicylic acid content in the leaves increased from 82.4 ng / g to 120.3 ng / g, an increase of 46%, providing a sufficient signal basis for SAR response and laying a good foundation for disease resistance.

[0086] Table 2: Comparison of salicylic acid content effects between this embodiment and the control example

[0087]

[0088] 3. Key enzyme activity and gene expression increased synchronously. Laccase (LAC) is a key enzyme in the final step of lignin monomer polymerization. The laccase activity increased from 1184 U / g to 3075 U / g, indicating that the formulation in this example can significantly increase the laccase activity of kiwifruit plants, thereby significantly activating the lignin polymerization step.

[0089] Table 3: Comparison of lignin polymerase (laccase) activity between this example and the control example

[0090]

[0091] 4. Enhanced lignin monomer synthesis capacity. The lignin monomer content of the plant continued to increase over 12 months, from 73.46 μg / mL to 147.68 μg / mL. This enhanced lignin monomer synthesis capacity directly strengthens the physical barrier of the cell wall.

[0092] Table 4: Comparison of lignin content effects between this example and the control example

[0093]

[0094] Based on the above embodiments, this invention, for the first time, incorporates a sulfur-succinic acid-Pseudomonas fluorescens ternary interaction system into the early spring nutrient management of kiwifruit, simultaneously achieving three-dimensional coupling of nutrition, signaling, and microorganisms within the rhizosphere microdomain. Field data clearly show that the endogenous salicylic acid signaling cascade in plants is continuously amplified, the activity of a key enzyme (laccase) in the lignin monomer synthesis pathway increases by 2.6 times, lignin deposition doubles, ultimately reducing the incidence of canker by 17-52%, with the effect remaining stable for at least 12 months, overcoming the bottleneck of the unstable prevention of traditional copper-based agents. This invention is the first to verify the operability of the rhizosphere immunity theory at the field scale, providing a paradigm for using fertilizers instead of pesticides to treat diseases in perennial fruit trees.

[0095] The technical terms, principles, or means related to the technical solutions of the present invention mentioned in the above embodiments, which are not described in detail above, are all well-known technologies or common practices that are known to those skilled in the art.

[0096] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A nutritional regulation method for activating systemic disease resistance in kiwifruit, the method targeting bacterial canker disease of kiwifruit caused by *Pseudomonas syringae* pv. *actinidiae* (Psa), characterized in that... The method includes constructing a ternary interaction system consisting of a sulfur source, succinic acid, and Pseudomonas fluorescens, and applying the ternary interaction system to early spring rhizosphere nutrient management of kiwifruit, thereby simultaneously achieving three-dimensional coupling of nutrient supply, signal activation, and microbial regulation within the rhizosphere microdomain. The sulfur source is used to initiate systemic disease resistance pathways in kiwifruit and activate the salicylic acid signaling pathway; the succinic acid is used to enhance the chemotaxis and biofilm formation ability of Pseudomonas fluorescens to promote its colonization in the rhizosphere, and further amplify the salicylic acid signaling pathway cascade reaction through the colonized Pseudomonas fluorescens; the sulfur source, succinic acid and Pseudomonas fluorescens synergistically upregulate the expression level of laccase gene in kiwifruit and enhance laccase activity. Meanwhile, the salicylic acid signaling pathway is continuously activated to promote lignin monomer synthesis. Highly active laccase, as a key enzyme in lignin monomer polymerization, accelerates the lignin monomer polymerization reaction, promotes the synthesis and deposition of lignin in the plant cell wall, continuously amplifies the endogenous disease resistance signal cascade in the plant, and induces stable systemic disease resistance in kiwifruit.

2. The nutritional regulation method for activating systemic disease resistance in kiwifruit according to claim 1, characterized in that, The sulfur source is elemental sulfur; the succinic acid has a purity of ≥98% and is a food-grade or agricultural-grade product; the fluorescent Pseudomonas includes similar strains isolated from the rhizosphere soil of sulfur-treated kiwifruit, exhibiting strong chemotaxis and high biofilm formation ability, with the effective viable count of the prepared functional bacterial solution ≥1×10⁻⁶. 8 CFU / mL.

3. The nutritional regulation method for activating systemic disease resistance in kiwifruit according to claim 1, characterized in that, Applying the aforementioned ternary interaction system to early spring rhizosphere nutrient management of kiwifruit involves preparing a special kiwifruit sprouting fertilizer using sulfur source, succinic acid, and Pseudomonas fluorescens as part of the raw materials, and then applying it to the kiwifruit.

4. The nutritional regulation method for activating systemic disease resistance in kiwifruit according to claim 1, characterized in that, The ternary interaction system employs a step-by-step application sequence optimization: first, sulfur source is mixed with well-rotted organic fertilizer and applied to the rhizosphere of kiwifruit for 7-10 days of pretreatment to improve the rhizosphere microenvironment and initiate the initial salicylic acid signal; then, succinic acid is mixed with Pseudomonas fluorescens bacterial solution and applied, so that succinic acid first provides suitable conditions for the colonization of the strain. After colonization, the strain synergistically amplifies the salicylic acid signaling pathway with the continuously released sulfur source, further improving the laccase gene expression efficiency and lignin deposition uniformity.

5. The nutritional regulation method for activating systemic disease resistance in kiwifruit according to claim 1, characterized in that, The sulfur source is modified by coating. The coating material is a biodegradable starch-chitosan composite membrane with a coating thickness controlled at 50-100 μm. The coating enables the sulfur source to be slowly released in the rhizosphere microdomain within 12 months. The release rate matches the colonization cycle of Pseudomonas fluorescens and the activation rhythm of the salicylic acid signaling pathway.

6. The nutritional regulation method for activating systemic disease resistance in kiwifruit according to claim 1, characterized in that, The *Pseudomonas fluorescens* bacterial suspension was pretreated with a low concentration of salicylic acid before use: the bacterial suspension was mixed with salicylic acid at a concentration of 10-20 ng / mL and incubated at 25-30℃ for 6-8 hours.

7. A special kiwifruit sprouting fertilizer for preventing and controlling bacterial canker, characterized in that, Its raw materials include a ternary interaction system composed of sulfur source, succinic acid and Pseudomonas fluorescens.

8. The kiwifruit-specific sprouting fertilizer for controlling bacterial canker according to claim 7, characterized in that, The raw material composition of this sprouting fertilizer, calculated by weight percentage, is as follows: 18-25 parts of 46% urea, 6-10 parts of 57% diammonium phosphate, 9-13 parts of granulated potassium sulfate, 3-7 parts of elemental sulfur, 10-20 parts of organic fertilizer, 0.5-2 parts of functional bacterial solution, and 0.5-2 parts of succinic acid; the functional bacterial solution is prepared from one or more fluorescent Pseudomonas bacteria.

9. The preparation method of the kiwifruit-specific sprouting fertilizer for controlling bacterial canker as described in claim 7, characterized in that, Includes the following steps: (1) The organic fertilizer is decomposed for 10–12 days, and the moisture content of the organic fertilizer after treatment is controlled to be 45–55% and the pH value is 6.0–6.8; (2) Add functional bacterial solution and succinic acid to the organic fertilizer treated in step (1), stir evenly and let stand for 24 hours to promote bacterial activation; (3) Mix 46% urea, 57% diammonium phosphate, granulated potassium sulfate and elemental sulfur, crush and sieve, with a sieve hole size ≤3mm, then dry to a moisture content ≤12% and complete packaging.

10. The preparation method of the kiwifruit-specific sprouting fertilizer for controlling bacterial canker according to claim 9, characterized in that, In step (1), the organic fertilizer is decomposed by composting, and the fermentation environment temperature is controlled at 25-35℃. The compost is turned over every 2-3 days. In step (3), the drying temperature is 50-60℃, and a low-temperature hot air drying method is used to avoid the volatilization of elemental sulfur and loss of nutrients caused by high temperature.

Images