Plant immune resistance inducer as well as preparation method and application thereof

Abstract

The invention belongs to but not limited to the technical field of microbial application, and discloses a plant immune resistance inducer as well as a preparation method and application thereof. Comprising an Aava fermentation extract, a surfactant, a defoaming agent, an anti-freezing agent, a thickening agent and a solvent, the Aava fermentation extract is a concentrate of an endophytic alternaria Aava fermentation filtrate, the surfactant is sodium dodecyl sulfate, the defoaming agent is tributyl phosphate, the antifreezing agent and the thickening agent are glycerol, and the solvent is water. The Aava fermentation extract disclosed by the invention is realized by stimulating autoimmunity of kiwi fruit plants, has no any sterilization effect on canker pathogens, and is not easy to generate drug resistance; and the compound has a certain broad spectrum, and has an induced resistance effect on gramineous plant wheat, rosaceae plant apple and solanaceae plant tobacco. When the Aava fermentation extract is used as a plant immune resistance inducer, the defense capability of plants can be improved, and the canker resistance of the plants can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to, but is not limited to, the technical field of microbial applications, and particularly relates to a plant immune inducer, a preparation method thereof, and applications thereof. Background Art

[0002] Plant diseases and insect pests are one of the major problems threatening the high-quality development of agriculture. Promoting the green prevention and control of diseases and insect pests has become a key measure to implement the concept of green development. In recent years, great efforts have been made to promote the green prevention and control of diseases and insect pests, which has played an important role in achieving zero growth of pesticides, reducing the use of chemical pesticides, improving the quality and safety of agricultural products, and promoting the green development of agriculture.

[0003] Plant immune inducers are a new practice in the field of green prevention and control of diseases and insect pests. As a new type of medicament, they mainly enhance the physiological functions of plants, improve the resistance of plants to pathogenic factors, and at the same time also have certain functions of promoting the growth of plant roots, stems and leaves and increasing crop yields. Due to their low toxicity, high environmental compatibility, and the property of not easily causing pathogens to develop resistance, they are gradually becoming an emerging practice in the field of plant protection and are being increasingly widely used in agricultural production. At present, the registered resistance induction factors of plant immune inducers mainly include sugars and organic acids. Research shows that biocontrol bacteria, proteins, and abiotic factors (ultraviolet rays, ozone, silica, etc.) are also important resistance induction factors that stimulate plant immunity and improve plant resistance. Among them, the fermentation products of some microbial strains also have the activity of stimulating plant immunity and are important active substances for creating plant immune inducers.

[0004] Existing resistance induction technologies basically activate plants during the seeding period or young bud period to induce the initiation of plant immunity. The plant immune inducer of the present invention can be combined with the appropriate application period for controlling kiwifruit canker disease, inhibit the accumulation of bacteria in kiwifruit plants in autumn, effectively control the occurrence of branch cankers in the following spring, and greatly reduce the application amount and application frequency of chemical pesticides. At the same time, most of the plant immune inducers reported for controlling canker disease are plant-derived components such as chelidonine and gall extract. The plant immune inducer of the present invention first uses the fermentation extract of microbial strain Aava to induce plant disease resistance and has further development and application value in agriculture.

[0005] There is currently no report on the use of the Aava fermentation extract as a plant immune inducer and for improving the defense ability of plants and the resistance of plants to canker disease. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the present invention provides a plant immune inducer, a preparation method thereof, and applications thereof.

[0007] The present invention is implemented as follows. A plant immune inducer comprises an Aava fermentation extract, a surfactant, an antifoaming agent, an antifreeze, a thickener and a solvent. The Aava fermentation extract is a concentrate of the fermentation filtrate of Alternaria alternata Aava. The Alternaria alternata Aava was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 28, 2021. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 21456. This strain is classified and named as Alternaria alternata.

[0008] Further, the surfactant is sodium dodecyl sulfonate, the antifoaming agent is tributyl phosphate, the antifreeze and the thickener are both glycerol, and the solvent is water.

[0009] Further, the mass percentages of each component are as follows: 0.5% of Aava fermentation extract, 0.25% of sodium dodecyl sulfonate, 0.3% of tributyl phosphate, 5% of glycerol, and the rest is solvent.

[0010] Further, the preparation method of the Aava fermentation extract specifically includes: culturing and activating Aava; inoculating the activated strain into a fermentation medium for fermentation culture to obtain a fermentation broth; performing suction filtration on the fermentation broth to separate out the liquid component; mixing the liquid component with n-butanol and rotary evaporating and concentrating; and finally drying the liquid component to obtain the Aava fermentation extract.

[0011] Another object of the present invention is to provide a preparation method of a plant immune inducer, which includes the following steps:

[0012] Step 1: First, quantify the solvent, and add the Aava fermentation extract into the solvent in a proportion determined by the amount of the solvent for dissolution.

[0013] Step 2: Add a quantified surfactant, antifreeze, antifoaming agent and thickener to the obtained dissolved medicament, and stir evenly to obtain the plant immune inducer.

[0014] Another object of the present invention is to provide an application of the plant immune inducer in improving the immune resistance of plants.

[0015] Another object of the present invention is to provide an application of the plant immune inducer in improving the resistance of plants to canker disease.

[0016] Further, the optimal application concentration of the plant immune inducer is 0.6 - 2.5 mg / mL.

[0017] Combined with the above technical solutions and the solved technical problems, the advantages and positive effects of the technical solution to be protected by the present invention are:

[0018] First, the Aava fermentation extract of the present invention is a concentrate of the fermentation filtrate of the biological strain Aava, which is an endophytic Alternaria Aava isolated from the bark of Chinese flowering crabapple.

[0019] When the Aava fermentation extract of the present invention is used as a plant immune elicitor, it can stimulate the up-regulation of resistance genes related to the salicylic acid pathway and ethylene pathway in kiwifruit plants, genes that induce the expression of plant pathogen-associated molecular patterns, and genes related to phenylalanine ammonia-lyase and superoxide dismutase, thereby improving the plant's defense ability and significantly enhancing the plant's resistance to canker disease.

[0020] The Aava fermentation extract of the present invention achieves the effect by stimulating the plant's own immunity, has no bactericidal effect on the canker pathogen, and is not prone to drug resistance; moreover, it has a certain broad spectrum and has an induction resistance effect on wheat of the Gramineae family, apple of the Rosaceae family, and tobacco of the Solanaceae family.

[0021] The raw materials required for the present invention are simple and easily available, the preparation method is simple, and compared with chemical fungicides on the market, it is green and environmentally friendly, safe for the environment, and has good prospects for development and utilization.

[0022] Second, as the creative auxiliary evidence of the claims of the present invention, it is also reflected in the following important aspects:

[0023] (1) The expected benefits and commercial value after the transformation of the technical solution of the present invention are:

[0024] The elicitor provided by the present invention can significantly reduce the economic losses caused by diseases, help reduce the damage of chemical residues to the ecological environment, protect the soil quality and water source safety, and conform to the development direction of modern green agriculture. At the same time, it helps to maintain the health and safety of agricultural products, meets the needs of consumers for green and organic foods, and can effectively enhance the market competitiveness and added value of agricultural products. The present invention uses the fermentation extract of the microbial strain Aava to prepare an immune elicitor, which has sustainability and renewability, conforms to the development trend of the biotechnology field, and provides a new way for the high-value utilization of microbial resources.

[0025] (2) The technical solution of the present invention fills the technical gaps in the domestic and international industries:

[0026] The elicitor provided by the present invention can be combined with the key technologies for the prevention and control of kiwifruit canker disease in the "two before and two after" periods, which is crucial for reducing the amount of chemical pesticides and increasing their efficiency, and for creating a common green prevention and control technology system guided by monitoring and early warning, based on immune elicitation, and centered on scientific pesticide application to ensure the healthy development of the industry. Description of the Drawings

[0027] Figure 1It is the antibacterial test result of the Aava fermentation extract provided by the embodiments of the present invention; A: sterile water control; B: Aava fermentation extract at 2 mg / mL; C: 5% amino-oligosaccharin diluted 500 times; D: 3% Zhongshengmycin WP diluted 80 times; E - H: different treatments of A - D under ultraviolet light;

[0028] Figure 2 It is the induction effect of the Aava fermentation extract provided by the embodiments of the present invention on the expression level of kiwifruit - related resistance genes;

[0029] Figure 3 It is the influence of the Aava fermentation extract provided by the embodiments of the present invention on the expansion and colonization of the canker pathogen in the leaf veins;

[0030] Figure 4 It is the prevention and control effect of the Aava fermentation extract provided by the embodiments of the present invention on kiwifruit canker;

[0031] Figure 5 It is the influence of different surfactants on the solution state and surface tension of the Aava fermentation extract solution provided by the embodiments of the present invention;

[0032] Figure 6 It is the influence of different antifreezes on the state of the Aava fermentation extract solution after 7 - day low - temperature storage provided by the embodiments of the present invention;

[0033] Figure 7 It is the prevention and control effect of different concentrations of plant immune elicitors on kiwifruit canker provided by the embodiments of the present invention;

[0034] Figure 8 It is the induction effect of the elicitor on the expression level of related resistance genes in wheat, tobacco, and apple provided by the embodiments of the present invention;

[0035] Figure 9 It is a schematic diagram of the influence of the Aava crude extract on the ROS content in kiwifruit provided by the embodiments of the present invention;

[0036] Figure 10 It is a schematic diagram of the influence of the Aava crude extract on the callose content in kiwifruit provided by the embodiments of the present invention;

[0037] Figure 11 It is a schematic diagram of the callose deposition in kiwifruit leaf tissues (observed by aniline blue staining) 24 hours after spraying the Aava fermentation broth extract solution (2 mg / mL) provided by the embodiments of the present invention;

[0038] Figure 12 It is a schematic diagram of the influence of the Aava crude extract on the ethylene content in kiwifruit provided by the embodiments of the present invention;

[0039] Figure 13It is a schematic diagram of the effect of Aava elicitor provided by an embodiment of the present invention on the total chlorophyll content of kiwifruit leaf tissue;

[0040] Figure 14 It is a schematic diagram of net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate provided by an embodiment of the present invention;

[0041] Figure 15 It is a schematic diagram of the cold resistance effect of Aava-induced kiwifruit branches provided by an embodiment of the present invention;

[0042] Figure 16 It is a schematic diagram of the effect of Aava extract on the MDA content of kiwifruit branches provided by an embodiment of the present invention. Detailed implementation manners

[0043] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] An embodiment of the present invention provides a plant immune elicitor, which includes Aava fermentation extract, surfactant, defoamer, antifreeze, thickener and solvent; the Aava fermentation extract is a concentrate of the fermentation filtrate of Alternaria endophytica Aava, and the Alternaria endophytica Aava was deposited on January 28, 2021 at the General Microbiology Center of the China Microbial Culture Collection Management Committee. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 21456. The strain is classified and named Alternaria alternata.

[0045] The surfactant is sodium dodecyl sulfonate, the defoamer is tributyl phosphate, the antifreeze and the thickener are both glycerol, and the solvent is water.

[0046] The mass percentages of each component are as follows: Aava fermentation extract 0.5%, sodium dodecyl sulfonate 0.25%, tributyl phosphate 0.3%, glycerol 5%, and the rest is solvent.

[0047] Example 1 Preparation of Aava fermentation extract

[0048] (1) Fermentation culture:

[0049] The Aava (Alternaria alternata var. alternata) strain preserved at 4°C was inoculated onto a flat PDA medium and cultured at 25°C for 7 days. Six mycelial discs (5 mm in diameter) were punched out and inoculated into a 1000 mL Erlenmeyer flask containing 400 mL of culture solution (PDB medium). After culturing at 25°C and 160 r / min for 10 days, the mycelium was filtered off through double-layer filter paper to obtain the Aava fermentation broth.

[0050] PDA medium formula: 200 g of potato, 20 g of glucose, 15 g of agar, 1000 mL of deionized water;

[0051] PDB medium formula: 200 g of potato, 20 g of glucose, 1000 mL of deionized water.

[0052] (2) Filtration and rotary evaporation:

[0053] The liquid component of the Aava fermentation broth was separated by filtration through double-circle qualitative filter paper, and the liquid component was mixed with n-butanol in a ratio of 7:3. The mixture was placed in a rotary evaporator and rotary evaporated at 55°C and 50 r / min. After taking it out, it was dried in an incubator at 65°C to obtain the Aava fermentation extract.

[0054] Example 2 Antibacterial effect of Aava fermentation extract against Pseudomonas syringae pv. actinidiae

[0055] (1) Measuring the inhibition zone by filter paper method:

[0056] 1 mL of the Pseudomonas syringae pv. actinidiae (Psa) bacterial solution (10 8 CFU / ml) was added to 1000 mL of solid LB medium that was heated and melted and cooled to 40°C. After thorough mixing, it was poured into plates and allowed to cool naturally. 5-mm filter paper discs were soaked in different treatment solutions for 5 min, taken out, air-dried, and placed in the center of the mixed-bacteria plates. The prepared plates were placed in an incubator at 28°C for 36 hours to observe the inhibition zone situation.

[0057] (2) Measuring the antibacterial effect by shaking culture method:

[0058] Different treatment solutions were mixed with LB medium at a ratio of 1:100 to obtain culture solutions. 20 μL of the Psa bacterial solution (10 8 CFU / ml) was pipetted into 2 mL of the culture solution, and the bacterial solution concentration was measured after culturing at 28°C and 180 r / min for 2 days.

[0059] (3) Test results:

[0060] As Figure 1As shown, Aava fermented extract has no antibacterial effect on Pseudomonas syringae pv. actinidiae.

[0061] Example 3 Inductive effect of Aava fermented extract on the resistance of kiwifruit plants

[0062] (1) Leaf treatment and sample collection:

[0063] Select healthy potted seedlings, spray with Aava extract solution (2 mg / mL), and use sterile water as the control (CK). Samples are collected at 0, 12, and 24 h after spraying. After collecting the leaves of each group, wrap them with tin foil, quickly freeze them in liquid nitrogen, and store them in a -80 °C refrigerator for later use.

[0064] (2) RNA extraction and cDNA synthesis:

[0065] Extract total RNA from samples at different time points according to the instructions of Huayueyang Polysaccharide and Polyphenol Plant Tissue RNA Extraction Kit. The concentration of the extracted RNA is measured by NanoDrop2000, and RNA with A260 / A280 and A260 / A230 values between 1.8 - 2.0 can be used for subsequent experiments. The reverse transcription method is carried out according to V on-step RT-gDNA digestionSuperMix for qPCR instructions.

[0066] (3) Primers for resistance-related genes and qRT-PCR:

[0067] Select kiwifruit resistance-related genes, with Actin as the internal reference gene. Primer synthesis is completed by Yangling Qingke Biotechnology Co., Ltd. Use 2xRealStar Green Power Mixture (#A311 - 05, GenStar), and according to the instructions, run on a real-time fluorescence quantitative PCR instrument (BioRad, Q5). The qPCR program is: pre-denaturation at 95 °C for 10 min; (denaturation at 95 °C for 15 s, annealing at 56 °C for 20 s, extension at 72 °C for 45 s) for 40 cycles. After the reaction, analyze the data. According to the Ct value of the gene, use the 2^(-ΔΔCT) method to calculate the relative expression level of the gene.

[0068] Table 1 Primer sequences of kiwifruit plant resistance-related genes

[0069]

[0070]

[0071] (4) Test results:

[0072] As Figure 2As shown in the figure, Aava fermentation extract can cause up-regulation of resistance genes PR1 and PR5 related to the SA signaling pathway in kiwifruit plants; genes CDPK and RBOH that induce PAMP expression; related enzyme genes PAL and CAT; upstream synthesis genes ACS2 and ACO7 in the ETH signaling pathway, and downstream transduction genes EIN2, EIN3, and EIL1 to varying degrees.

[0073] Example 4 Effect of Aava fermentation extract on the colonization and spread of Pseudomonas syringae pv. actinidiae in kiwifruit leaf veins

[0074] (1) Leaf treatment and sample collection:

[0075] Select healthy potted seedlings, spray with Aava extract solution (2 mg / mL), and use sterile water as the control (CK). Samples were collected 24 h after spraying. Disinfect the leaf surface, disinfect with 0.6% NaClO solution for 5 min (note to avoid damaging the leaves during the experiment), rinse with sterile water and dry the residual moisture on the leaf surface with filter paper. Pierce the main leaf vein with a sterile needle, and drop 10 μL of Psa bacterial solution (10 4 CFU / ml) at the wound, with sterile water as the control. Keep it moist and place it in an artificial climate chamber. The culture conditions of the artificial climate chamber are photoperiod L / D: 16 h / 8 h; day and night temperature: 16 °C; relative humidity: 95%. Observe and record the spread of the pathogen in the leaf veins every 7 days.

[0076] (2) Test results:

[0077] As Figure 3 shown, Aava fermentation extract can significantly reduce the colonization amount and spread rate of Psa in kiwifruit leaf veins.

[0078] Example 5 Control effect of Aava fermentation extract on kiwifruit canker

[0079] (1) Leaf disc vacuum infiltration inoculation:

[0080] Select healthy potted seedlings, spray with Aava extract solution (2 mg / mL), and use sterile water as the control (CK). Samples were collected 24 h after spraying. Disinfect the leaf surface, disinfect with 0.6% NaClO solution for 5 min (note to avoid damaging the leaves during the experiment), rinse with sterile water and dry the residual moisture on the leaf surface with filter paper. Use a sterile punch (11 mm in diameter) to make leaf discs, avoiding the main leaf vein; place the leaf discs in Psa bacterial solution (10 4(CFU / ml), using sterile water as a control, perform vacuum infiltration (until the leaf discs are completely submerged in the bacterial solution and there are no air bubbles); wash three times with sterile water to remove the mucus exuded by the leaf discs, and finally use sterilized filter paper to absorb the excess water on the leaf discs, place them on a 0.8% water agar plate, and then place them in an artificial climate chamber. The culture conditions of the artificial climate chamber are light cycle L / D: 16h / 8h; day and night temperature: 16°C; relative humidity: 95%. Observe the expansion of the pathogen in the leaf discs after 5 days.

[0081] (2) Inoculation on wounded detached branches:

[0082] Select healthy potted seedlings, spray with Aava extract solution (2mg / mL), using sterile water as a control (CK), and sample 24h after spraying. Disinfect the collected healthy detached branches with 0.6% NaClO solution for 15 minutes, then repeatedly wash with sterilized distilled water for about 15 minutes, repeat 3 to 4 times until there is no pungent smell on the branch surface, and wait for the branch to dry; cut into short branches about 15cm long, seal both ends with paraffin; artificially create wounds (cut with a single-sided blade, 1mm wide, cut to the phloem), drop 10 μL of Psa bacterial solution (10 8 CFU / ml) on the wound, using the branch with sterile water dropped as a control; after the bacterial solution and sterile water penetrate into the branch, place it in a tray, add an appropriate amount of sterile water to the tray, cover it with a film to keep it moist, and then place it in an artificial climate chamber. The culture conditions of the artificial climate chamber are light cycle L / D: 16h / 8h; day and night temperature: 16°C; relative humidity: 95%. Observe the expansion of the pathogen in the leaf discs after 30 days.

[0083] (3) Test results:

[0084] As Figure 4 shown, Aava fermentation extract has a certain control effect on kiwifruit canker, with a leaf control effect of 85.36% and a branch control effect of 48.06%.

[0085] Example 6 Formula screening and quality evaluation of plant immune elicitors

[0086] (1) Screening of surfactants:

[0087] The alternative surfactants are sodium dodecyl sulfonate, sodium dodecyl sulfate, sodium 1-octane sulfonate, and fatty alcohol polyoxyethylene ether. The best surfactant is selected as sodium dodecyl sulfonate through the solubility and surface tension of the surfactant in the Aava fermentation extract solution, and the best addition mass fraction is 0.25%. The specific results are shown in Figure 5 and Tables 2, 3, and 4:

[0088] Table 2 Screening of surfactant types

[0089]

[0090] Table 3 Surface Tension at Different Concentrations of Sodium Dodecyl Sulfonate

[0091]

[0092] Table 4 Surface Tension at Different Concentrations of Sodium 1 - Octanesulfonate

[0093]

[0094]

[0095] (2) Screening of Defoamers:

[0096] The alternative defoamers are tributyl phosphate, methyl silicone oil, Tween 80, and polyethylene glycol 200 oil ether. The defoamers are screened by measuring the persistent foaming property (performed according to Standard GB / T 28137 - 2011). At room temperature, add standard hard water (342 mg / L) to a 250 mL stoppered graduated cylinder up to the 180 mL graduation line; place the graduated cylinder on a balance, add 1.0 g of the sample, and then add hard water up to the graduation line at a distance of (9 ± 0.1) cm from the bottom of the stopper of the graduated cylinder; cover the stopper, and with the middle of the graduated cylinder as the center, invert it 180 times up and down 30 times, 2 s each time; record the foam volume at 1 min. Finally, tributyl phosphate is selected as the best defoamer, and the optimal addition mass fraction is 0.3%. The specific results are shown in Table 5:

[0097] Table 5 Persistent Foaming Property of Different Kinds of Defoamers

[0098]

[0099]

[0100] (3) Screening of Antifreeze Agents:

[0101] The alternative antifreeze agents are ethylene glycol, glycerol, propylene glycol, and urea. The antifreeze agents are screened by observing the stability of the Aava fermentation extract solution after storage at 0 °C for 1 h and 7 d with different concentrations of antifreeze agents added. (Performed according to the Low - Temperature Stability Determination Method of Standard GB / T19137 - 2003), and the suitable antifreeze agents are 5% - 10% ethylene glycol and glycerol. The specific results are shown in Figure 6 and Table 6:

[0102] Table 6 Low - Temperature Stability of Different Kinds of Antifreeze Agents

[0103]

[0104]

[0105] (4) Screening of thickeners:

[0106] The alternative thickeners are polyvinyl alcohol, sodium carboxymethyl cellulose, magnesium aluminum silicate, xanthan gum, and glycerol. By observing the viscosity of the Aava fermentation extract solution with different concentrations of thickeners added, the appropriate antifreeze agent was screened out as 2% glycerol.

[0107] The specific results are shown in Table 7:

[0108] Table 7 Determination results of different types of thickeners

[0109]

[0110] In summary, the optimal formula of the plant immune elicitor is 0.5% Aava fermentation extract, 0.25% sodium dodecyl sulfate, 0.3% tributyl phosphate, and 5% glycerol. The quality of the plant immune elicitor was determined (performed according to the standard HG / 2467.1 - 2467.20 - 2003), and the specific results are shown in Table 8:

[0111] Table 8 Quality determination results of the plant immune elicitor

[0112]

[0113] Example 7 Evaluation of the control effect of the plant immune elicitor against kiwifruit canker and screening of application concentration

[0114] (1) Treatment of kiwifruit samples:

[0115] Samples were taken 24 hours after spraying the plant immune elicitor at different concentrations, and the treatment method was the same as in Example 5.

[0116] (2) Test results:

[0117] As Figure 7 shown, when the plant immune elicitor was applied at a dilution of 2 to 8 times (corresponding concentration of 0.6 - 2.5 mg / mL), no obvious disease spots were observed on the leaves, and the control effect on the branches was 72.12%, with the best application effect, which is the recommended application concentration of this plant immune elicitor.

[0118] Example 8 Evaluation of the broad-spectrum elicitor effect of the plant immune elicitor

[0119] (1) Treatment of plant samples

[0120] Select the gramineous plant wheat, the rosaceous plant apple, and the solanaceous plant tobacco as the test plants. Samples were taken 24 hours after spraying the plant immune elicitor (2 mg / mL), and the treatment method was the same as in Example 4.

[0121] (2) Test results

[0122] As Figure 8As shown, after treatment with the elicitor, the expression levels of most immune-related genes in apple, wheat and tobacco were upregulated to varying degrees, indicating that the elicitor has a certain broad-spectrum inducing function.

[0123] 1. Specific application fields or related products of the present invention.

[0124] Example 9 Application effect of plant immune inducer and compound application effect

[0125] (1) Plant sample processing

[0126] Healthy kiwifruit potted seedlings (variety: Hongyang) were selected, and 24 hours after the application of different treatment group agents (application method: uniform spray), the leaves of different treatment groups were picked by the five-point sampling method, and the leaves were inoculated with bacteria (treatment method is the same as Example 5), and placed in an artificial climate box to keep moisture. The culture conditions of the artificial climate incubator are light cycle L / D: 16h / 8h; day and night temperature: 16℃; relative humidity: 95%. The incidence of ulcer disease was observed after 5 days. The details of different treatment agents are shown in Table 9:

[0127] Table 9

[0128]

[0129] Note: A is Aava plant immune inducer (0.6 mg / mL); B is 3% zhongshengmycin wettable powder (1600 times dilution), Dongguan Redefeng Biotechnology Co., Ltd.

[0130] (2) Test results

[0131] As shown in Table 10, Aava immune inducer has a good preventive effect on kiwifruit canker, with a preventive effect of 84.35%. At the same time, the inducer and the common antibiotics for canker prevention and control all show additive effects in various compound ratios. Among them, the combination ratio of 6:4 shows a synergistic effect, with a preventive effect of 98.2%, showing good application potential.

[0132] Table 10

[0133]

[0134]

[0135] 2. Relevant evidence of the technical effects obtained by the embodiments of the present invention.

[0136] Plant immune inducers are gradually becoming an emerging practice in the field of plant protection due to their low toxicity, high environmental compatibility and low resistance to pathogens.

[0137] In the plant immune elicitor of the present invention, the fermentation extract of the plant endogenous microbial strain Aava is used as the active substance to stimulate the plant's own immune regulation mechanism, which can significantly up-regulate the expression of genes related to the ethylene pathway, salicylic acid pathway, and resistance defense enzymes in kiwifruit plants.

[0138] The plant immune elicitor in the present invention has a significant prevention and control effect on kiwifruit canker. The control effect on leaves reaches 84.35%, and the control effect on branches reaches 72.12%.

[0139] The plant immune elicitor in the present invention has a certain broad-spectrum elicitor effect and has a certain induced resistance effect on test plants such as apple in Rosaceae, wheat in Gramineae, and tobacco in Solanaceae.

[0140] In addition, the active ingredient Aava fermentation extract of the plant immune elicitor in the present invention can stimulate the classic immune response of kiwifruit - the burst of reactive oxygen species (ROS) and the deposition of callose, as Figure 9 、 Figure 10 、 Figure 11 shown.

[0141] In addition, the active ingredient Aava fermentation extract of the plant immune elicitor in the present invention can cause a significant increase in the ETH content of kiwifruit plants, which can be increased by up to 1.84 times within 72 hours; under the condition of the most suitable temperature of 16°C for the occurrence of kiwifruit canker, it can significantly delay the decrease in the ETH content of kiwifruit plants, as Figure 12 shown.

[0142] In addition, the plant immune elicitor in the present invention can improve the growth potential of kiwifruit trees and has a certain promoting effect on gas exchange function and cold resistance. After treatment with the Aava immune elicitor, the relative chlorophyll content of leaves shows an upward trend within 5 days. After treatment for 48 hours, the stomatal conductance of leaves increases, the intercellular CO2 concentration decreases, and the net photosynthetic rate and transpiration rate are both enhanced to a certain extent; at the same time, after treatment for 24 hours, the degree of electrolyte leakage of kiwifruit branches shows a downward trend at different temperatures, with the highest decrease of 13.56%. The accumulation rate of malondialdehyde in branches slows down within 72 hours of treatment, and membrane lipid peroxidation is inhibited, and the overall cold resistance shows a certain degree of enhancement, as Figure 13 、 Figure 14 、 Figure 15 、 Figure 16 shown.

[0143] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention shall be covered by the protection scope of the present invention.

Claims

1. A plant immune inducer, characterized in that, It includes Aava fermentation extract, surfactant, defoamer, antifreeze, thickener and solvent; the Aava fermentation extract is a concentrate of the fermentation filtrate of Alternaria alternata Aava. The Alternaria alternata Aava was deposited on January 28, 2021 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 21456. This strain is classified and named as Alternaria alternata.

2. The plant immune inducer according to claim 1, characterized in that, The surfactant is sodium dodecyl sulfate, the defoamer is tributyl phosphate, the antifreeze and the thickener are both glycerol, and the solvent is water.

3. The plant immune inducer according to claim 2, wherein, The mass percentages of each component are as follows: Aava fermentation extract 0.5%, sodium dodecyl sulfate 0.25%, tributyl phosphate 0.3%, glycerol 5%, and the rest is solvent.

4. The plant immunity elicitor according to claim 1, characterized in that, The preparation method of the Aava fermentation extract specifically includes: culturing and activating Aava; inoculating the activated strain into a fermentation medium for fermentation culture to obtain a fermentation broth; performing suction filtration on the fermentation broth to separate out the liquid component; mixing the liquid component with n-butanol and rotary evaporating and concentrating; and finally drying the liquid component to obtain the Aava fermentation extract.

5. A preparation method of the plant immune inducer as described in any one of claims 1 to 4, characterized in that, It includes the following steps: Step 1: First, quantify the solvent, and add the Aava fermentation extract into the solvent at a proportion determined by the solvent amount for dissolution. Step 2: Add a quantified surfactant, antifreeze, defoamer and thickener to the obtained dissolved medicament, and stir evenly to obtain a plant immune elicitor.

6. Use of a plant immune elicitor according to any one of claims 1 to 4 in enhancing the immune resistance of plants.

7. Use of a plant immune elicitor according to any one of claims 1 to 4 in enhancing the resistance of plants to canker disease.

8. The application according to claim 7, wherein The optimal application concentration of the plant immune elicitor is 0.6 - 2.5 mg / mL.

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