A method for preparing and applying a dextran heptahydrate

Abstract

This invention relates to a method for preparing and applying heptacaprans, belonging to the field of natural product synthesis technology. The preparation method of this invention is a completely different route compared to previously reported heptacapran preparation methods. Each reaction step has been optimized, with the yields of each step involving the glucose tetrasaccharide donor, glucose trisaccharide acceptor, and heptacaprans exceeding 90%, a significant improvement compared to the previous method's yield of around 70-80%, representing a substantial technological advancement. The heptacaprans of this invention is effective in controlling strawberry anthracnose and tomato bacterial wilt, and its effectiveness against these diseases is superior to commercially available amino oligosaccharides, demonstrating significant technological progress. The heptacaprans of this invention is ineffective against powdery mildew in cucurbits, indicating that it is not effective against all plant diseases. This invention was obtained unexpectedly through extensive experimentation, demonstrating significant efficacy against strawberry anthracnose and tomato bacterial wilt.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying dextran heptahydrate, belonging to the field of natural product synthesis technology. Background Technology

[0002] The main chain of glucose oligosaccharides is linked by 1→6β, and the side chains are linked by 1→3β. Glucose oligosaccharides are a novel type of biopesticide that can stimulate the plant's immune system to prevent and control diseases and pests. While not toxic itself, it can stimulate the development of systemic acquired resistance in plants, stimulating their internal immune mechanisms to combat and prevent disease. Because glucose oligosaccharides have a significant effect on activating the plant's immune system and regulating plant growth, and because they do not pollute the environment and are harmless to human health, they are poised to replace pesticides and plant growth hormones, changing the future industrial structure of pesticides and pesticide formulations, and becoming a new direction for pesticide development.

[0003] Although it is mentioned that glucan has the potential to stimulate the plant immune system, there is no literature on which specific plants or diseases glucan is specifically applied to, which plant diseases it is effective against, or how much better it is than commercially available agents.

[0004] The reported methods for synthesizing glucose oligosaccharides are all quite complex and have low raw material utilization rates, which increases production costs and is not conducive to large-scale industrial production.

[0005] The previously reported method for preparing dextran heptadecanoate in patent 201810631305.0 requires the use of monosaccharide 5 (4,6-benzyl-2,3-di-O-benzoylglucose trichloroacetylimine ester), which is difficult to synthesize. The first step involves the reaction of glucose with benzaldehyde dimethyl acetal, which is expensive, resulting in a low yield. The third step, the removal of the 1-position benzoyl group, is also difficult and yields low results. Both of these reactions are challenging problems in saccharochemistry, leading to low yields and high costs in the synthesis of monosaccharide 5. Summary of the Invention

[0006] The preparation method of this invention is completely different from the previously reported methods for preparing dextran heptaose. Compared to previous synthesis steps, each reaction step has been optimized. The synthesis yields of each step involving the glucose tetrasaccharide donor, glucose trisaccharide acceptor, and dextran heptaose are all above 90%, a significant improvement compared to the previous method's yield of around 70-80%, representing a significant technological advancement. The dextran heptaose of this invention is effective in controlling strawberry anthracnose and tomato bacterial wilt, and its effectiveness against these diseases is better than that of commercially available amino oligosaccharides, demonstrating significant technological progress. The dextran heptaose of this invention is ineffective against powdery mildew in cucurbits, indicating that it is not effective against all plant diseases. This invention also unexpectedly yielded significant results against strawberry anthracnose and tomato bacterial wilt through extensive experimentation.

[0007] The technical problem solved by this invention is the application of dextran heptahydrate, the structural formula of which is as follows:

[0008] The aforementioned glucan is used to prepare agents for treating strawberry anthracnose and tomato bacterial wilt.

[0009] The EC50 value for exogenous application of glucan for the control of strawberry anthracnose was 6.072 mg / kg.

[0010] The EC50 value for exogenous application of glucan to control bacterial wilt in tomatoes was 4.220 mg / kg.

[0011] Beneficial effects:

[0012] The preparation method of glucan heptaose of the present invention is a completely different preparation route compared with the previously reported preparation methods. Compared with the previous synthesis steps, each reaction step has now been optimized. The synthesis yield of each step of the glucose tetrasaccharide donor, glucose trisaccharide acceptor, and glucan heptaose is above 90%, which is a significant improvement compared with the synthesis yield of about 70-80% of the previous method, and has significant technical progress.

[0013] The heptacapran of this invention is effective in controlling strawberry anthracnose and tomato bacterial wilt, and its effect on controlling these diseases is better than that of commercially available amino oligosaccharides. The control effect of concentrated heptacapran against strawberry anthracnose fungus is similar at a concentration of 10 mg / kg to that of 100 mg / kg amino oligosaccharides. (50 mg·L⁻¹) -1 and 20 mg·L -1 Dextran showed the best induction of disease resistance in tomatoes, with relative control efficacies of 65.47% and 62.39% respectively 10 days after inoculation. This was significantly higher than the control fungicide amino oligosaccharide 100 mg·L⁻¹. -1There was no significant difference in relative efficacy (63.93%), and the 20 mg·L⁻¹ efficacy was significantly lower. -1 The treatment had a relatively long induction period, and the relative efficacy was still 60.26% 13 days after inoculation, which was higher than the relative efficacy of the fungicide control treatment (47.10%) under the same conditions, demonstrating significant technological progress.

[0014] The glucan of the present invention is ineffective against powdery mildew in cucurbits, indicating that the glucan of the present invention is not effective against all plant diseases. The present invention was obtained unexpectedly through a large number of experiments and showed significant effects against anthracnose in strawberries and bacterial wilt in tomatoes. Attached Figure Description

[0015] The present invention will be further described below with reference to the accompanying drawings.

[0016] Figure 1 The effect of the experimental agent on the phenotype of strawberry anthracnose after disease onset.

[0017] Figure 2 Effects of the test agent on the control of strawberry anthracnose

[0018] Figure 3 The effect of the test agent on the EC50 value of strawberry anthracnose

[0019] Figure 4 Phenotypic changes in tomato plants after bacterial wilt

[0020] Figure 5 The induction of bacterial resistance to tomato wilt by glucan: A, B: 10 days after inoculation; C, D: 13 days after inoculation.

[0021] Figure 6 glucan affects the EC50 of bacterial wilt in tomatoes. Detailed Implementation

[0022] Example 1

[0023] Preparation of glucose tetrasaccharide donors:

[0024] 1) Dissolve 4.396 g, 5 mmol monosaccharide 1 in 40 ml dichloromethane to obtain solution A; dissolve 1.472 g, 5 mmol monosaccharide 2 in 10 ml dichloromethane to obtain solution B; mix solution A and B to obtain solution C; add 90 μL, 0.5 mmol TMSOTf catalyst to solution C, then add 4A molecular sieve, and react at 25 °C for 3 h. Thin-layer chromatography analysis showed that the reaction was complete. Filter, evaporate the solvent under reduced pressure, separate by column chromatography, and elute with ethyl acetate / cyclohexane (volume ratio 1 / 3) as the eluent. Collect the corresponding components to obtain pure disaccharide 3 with a yield of 92%.

[0025]

[0026] 2) Dissolve 5.056 g of 5 mmol disaccharide 3 in 50 mL of 80% acetic acid aqueous solution, and react at 25 °C for 1 minute. -1 Thin-layer chromatography (TLC) analysis after 0.5 h indicated the reaction was complete. The solvent was removed under reduced pressure, and the mixture was separated by column chromatography using ethyl acetate / cyclohexane (1 / 2 v / v) as the eluent. The fractions were collected to obtain pure disaccharide 4. Different acidities affected the yield. The yield was 93% with 70% acetic acid aqueous solution, 95% with 90% acetic acid aqueous solution, and the highest yield (98%) with 80% acetic acid aqueous solution.

[0027]

[0028] 3) Dissolve 3.705 g (5 mmol) of monosaccharide 5 in 30 ml of dichloromethane to obtain solution A; dissolve 1.922 g (2.5 mmol) of disaccharide 4 in 20 ml of dichloromethane to obtain solution B. Mix solutions A and B to obtain solution C; add 90 μL (0.5 mmol) of TMSOTf catalyst to solution C, then add 4A molecular sieve. After reacting at 25 °C for 4 h, thin-layer chromatography analysis showed that the reaction was complete. Filter, evaporate the solvent under reduced pressure, separate by column chromatography, and elute with ethyl acetate / cyclohexane (volume ratio 1 / 2.5). Collect the corresponding fractions to obtain pure tetrasaccharide 6. The yield varies with different reaction concentrations. When the material is at 5 mmol, the yield is 88% when monosaccharide 5 is dissolved in 20 ml of dichloromethane. The yield is 87% when monosaccharide 5 is dissolved in 40 ml of dichloromethane. The highest yield (91%) is obtained when monosaccharide 5 is dissolved in 30 ml of dichloromethane.

[0029]

[0030] 4) Dissolve 3.852 g and 2 mmol of tetrasaccharide 6 in 50 ml of 1% hydrochloric acid-methanol solution and react at 25 °C for 2-2.5 h. Thin-layer chromatography analysis showed that the reaction was complete. The solvent was removed by vacuum distillation, and the mixture was separated by column chromatography. The eluent was ethyl acetate / cyclohexane (volume ratio 1 / 3). The corresponding components were collected to obtain pure tetrasaccharide 7 with a yield of 96%.

[0031] 5) Dissolve 3.623 g and 2 mmol of tetrasaccharide 7 in 30 mL of dichloromethane, add 0.4 mL of 4 mmol of trichloroacetonitrile and 0.552 g of 4 mmol of potassium carbonate, stir at 25 °C for 5 h. Thin-layer chromatography analysis showed that the reaction was complete. The solvent was evaporated under reduced pressure, and the mixture was separated by column chromatography. Ethyl acetate / cyclohexane (volume ratio 1 / 2.5) was used as the eluent, and the corresponding fractions were collected to obtain the tetrasaccharide 8 donor with a yield of 98%.

[0032]

[0033]

[0034] Preparation of glucosamine receptors:

[0035] 1) Dissolve 5.056 g, 5 mmol of disaccharide 3 in 50 ml of dichloromethane to obtain solution A; dissolve 3.705 g, 5 mmol of monosaccharide 5 in 10 ml of dichloromethane to obtain solution B; mix solution A and B to obtain solution C; add 90 μL, 0.5 mmol of TMSOTf catalyst to solution C, then add 4A molecular sieve, and react at 25 °C for 3 h. Thin-layer chromatography analysis showed that the reaction was complete. Filter, evaporate the solvent under reduced pressure, separate by column chromatography, and elute with ethyl acetate / cyclohexane (volume ratio 1 / 3) as the eluent. Collect the corresponding components to obtain pure trisaccharide 9 with a yield of 91%.

[0036] 2) Dissolve 3.180 g of 2 mmol of trisaccharide 9 in 30 mL of 80% acetic acid aqueous solution, and react at 25 °C for 1 minute. -1 After 0.5 h, thin-layer chromatography analysis showed that the reaction was complete. The solvent was evaporated under reduced pressure, and the mixture was separated by column chromatography. The eluent was ethyl acetate / cyclohexane (volume ratio 1 / 3). The corresponding components were collected to obtain pure trisaccharide 10 acceptor with a yield of 98%.

[0037]

[0038]

[0039] Synthesis of the target compound:

[0040] 1) 2.695 g of 2 mmol glucosamine trisaccharide 10 acceptor, 3.9123 g of 2 mmol glucosamine tetrasaccharide 8 acceptor, and molecular sieve were dissolved in 50 mL of anhydrous dichloromethane and stirred for 1.5 h under nitrogen protection. Then, 72 μL of 0.40 mmol TMSOTf catalyst was added dropwise, and the reaction was carried out at 25 °C for 4.5 h. Thin-layer chromatography analysis showed that the reaction was complete. The mixture was filtered, the solvent was evaporated under reduced pressure, and the mixture was separated by column chromatography. Eluent was ethyl acetate / cyclohexane (1 / 1.8) and the corresponding fractions were collected to obtain pure heptaose 11 with a yield of 86%.

[0041] 2) 6.282 g (2 mmol) of heptaose 11 was dissolved in 35 mL of 1% hydrochloric acid-methanol solution and reacted at 25 °C for 2-2.5 h. Thin-layer chromatography analysis showed that the reaction was complete. The solvent was removed under reduced pressure, and the mixture was separated by column chromatography using ethyl acetate / cyclohexane (1 / 1.8) as the eluent. The corresponding fractions were collected to obtain pure heptaose 12, with a yield of 95% under these conditions. Different concentrations of hydrochloric acid affected the yield; the yield was 92% with 0.5% hydrochloric acid-methanol solution and 93% with 1.5% hydrochloric acid-methanol solution.

[0042] 3) Dissolve 6.054 g and 2 mmol of heptaose 12 in a saturated sodium methoxide solution, stir for 78 h, concentrate, and then separate by dextran gel LH-20 (methanol) column to obtain the target product with a yield of 83%. Data of the target product: ESMS m / z 1184{M+NH4}, FW=1166.39.

[0043] The synthesis reaction formula is as follows:

[0044]

[0045]

[0046]

[0047]

[0048] Example 2

[0049] Indoor activity assay of glucan against *Colletotrichum acutatum*, the causal agent of strawberry anthracnose.

[0050] 1. Experimental Objective

[0051] Strawberry anthracnose, caused by *Colletotrichum acutatum*, is a common disease in greenhouse strawberry cultivation. This pathogen can infect various organs of strawberries, including roots, petioles, leaves, flowers, fruits, and runners, causing a series of symptoms such as root rot, leaf spot, fruit rot, and decreased reproductive capacity, severely limiting the development of the strawberry industry. Following the People's Republic of China Agricultural Industry Standard NY_T 1156.14-2008, the effect of glucan on the indoor activity of the strawberry anthracnose pathogen was determined using a pot method.

[0052] 2. Experimental Materials

[0053] Unless otherwise specified, all reagents used in this method are of analytical grade; the water used is distilled water.

[0054] 2.1 Biological test materials

[0055] The pathogen tested was *Colletotrichum acutatum* Simmonds strain. The crop tested was *Strawberry 'Nine-Nine'*, which was potted and cultured until it had 2-4 true leaves. The samples were numbered for future reference.

[0056] 2.2 Instruments and Equipment

[0057] Common laboratory equipment includes: electronic balance (sensitivity 0.1 mg), spray apparatus, artificial climate chamber, pipettes or pipettes, etc.

[0058] 2.3 Test and control reagents

[0059] The mixture consisted of 95% dextran technical grade (prepared in Example 1) and amino oligosaccharide technical grade.

[0060] 3. Experimental Procedure

[0061] 3.1 Preparation of spore suspension

[0062] Strawberry anthracnose fungus was cultured on PDA medium. After spores were produced, the spores were washed off with sterile water and filtered through double-layer gauze to prepare a suspension with a concentration of 1×10⁵ spores / mL for later use.

[0063] 3.2 Drug Preparation

[0064] Dextran and amino oligosaccharide were dissolved and diluted directly with distilled water, with 0.1% Tween-80 or other suitable surfactant added. Based on the pharmaceutical activity, seven series of mass concentrations were set for dextran: 0.1, 1, 5, 10, 20, 50, and 100 mg / kg. For amino oligosaccharide, 100 mg / kg was used as the working concentration.

[0065] 3.3 Chemical treatment

[0066] Spray the solution evenly onto the leaves until completely wet, and allow the solution to air dry naturally before use. Each treatment consists of 3 pots, with 4 replicates. A negative control is provided, consisting only of water without the active ingredient, and a positive control is provided, consisting of an aqueous solution containing 100 mg / kg of amino oligosaccharide.

[0067] 3.4 Inoculation and Culture

[0068] After the potted plants reach the seedling stage, induce them with the fungicide once every 7 days, for a total of 3 inductions. Inoculate with the pathogen 24 hours after the last induction. Use a micro-sampler to take 10 μL of spore suspension and inoculate it onto the upper surface of the leaves, with no fewer than 30 inoculation points per treatment. After inoculation, transfer them to a humidity chamber (relative humidity 95%–100%) and incubate in the dark for 24 hours, then continue incubation at 22℃–27℃, light intensity 20000 Lux, and humidity 80%–90%.

[0069] 3.5 Survey

[0070] When the incidence rate in the negative control reaches 50% or more, the diameter of the lesion is measured once using a vernier caliper with the cross-sectional perpendicular method, and the average value is taken. The unit is millimeters (mm).

[0071] 4. Data Statistics and Analysis

[0072] 4.1 Calculation Method

[0073] Based on the survey data, calculate the prevention and control effect using the formula below, expressed as a percentage (%), and retain two decimal places in the calculation result.

[0074]

[0075] In the formula:

[0076] P – Prevention and control effect, expressed as a percentage (%).

[0077] D0—Diameter of negative control lesions, in millimeters (mm);

[0078] D1 – Diameter of the lesion treated with the agent, in millimeters (mm).

[0079] 4.2 Statistical Analysis

[0080] The control effect was plotted using GraphPad Prism 8 software, and regression analysis was performed on the logarithmic values ​​of pesticide concentrations and control effect values ​​to calculate the EC50 value of each pesticide.

[0081] 5. Results Analysis

[0082] Combining the phenotypic observation in Figure 1 and the control effect analysis in Figure 2, it was found that the control effect against strawberry anthracnose was enhanced with the increase of glucan concentration. The concentration of glucan at 10 mg / kg was the lowest concentration with an effect similar to that of amino oligosaccharide at 100 mg / kg.

[0083] Analysis revealed that the EC50 value of exogenous spraying of glucan for controlling strawberry anthracnose was 6.072 mg / kg (Figure 3).

[0084] 6. Conclusion

[0085] The experimental results showed that the indoor activity test of 95% glucan technical material against strawberry anthracnose fungus using the pot method yielded an EC50 value of 6.072 mg / kg.

[0086] Example 3

[0087] The effect of glucan on the prevention and control of bacterial wilt in tomatoes

[0088] This experiment referenced the following standards: NY / T 1156.16-2008 "Indoor Bioassay Test Guidelines for Pesticides - Fungicides Part 16: Turbidity Method for Inhibition of Bacterial Growth" and NY / T 1464.32-2010 "Field Efficacy Test Guidelines for Pesticides Part 32: Control of Bacterial Wilt in Tomatoes by Fungicides".

[0089] 1. Instruments and Equipment

[0090] Standard laboratory equipment includes: electronic balance (sensitivity 0.1 mg), spray apparatus, artificial climate chamber, pipettes or pipettes, etc.

[0091] 2. Materials and Methods

[0092] 2.1. Experimental Materials

[0093] 2.1.1. Biological test materials

[0094] In this study, Ralstonia solanacearum physiological race 1 was tested on tomato variety L-404, which was bred by Liaoning Academy of Agricultural Sciences.

[0095] 2.1.2. Test reagents

[0096] 95% glucan-7-glucan raw material.

[0097] 2.1.3. Control reagent

[0098] Water and amino oligosaccharide raw materials

[0099] 2.1.4. Culture medium and solution:

[0100] (1) NB medium: 3.0 g beef extract; 1.0 g yeast extract; 5.0 g peptone; 10.0 g glucose; ddH2O to 1.0 L; pH 7.0-7.2.

[0101] (2) NA medium: 3.0 g beef extract; 1.0 g yeast extract; 5.0 g peptone; 10.0 g glucose; 15.0 g agar powder; ddH2O to a final volume of 1.0 L; pH 7.0-7.2.

[0102] (3) TTC stock solution: Prepare a 1% solution with distilled water and sterilize by filtration through a 0.22 μm filter membrane. Dispense and store at 4℃. Before pouring NA medium into plates (cool to about 45℃), add 0.5 mL of 1% TTC to every 100 mL of medium.

[0103] (4) 10 mM MgCl2 solution: Prepare 1 M MgCl2 solution with distilled water, then autoclave at 121℃ for 20 min, store at room temperature, and dilute with ddH2O before use.

[0104] 2.2. Experimental Methods

[0105] 2.2.1. Tomato Seed Pretreatment

[0106] (1) In this study, tomato seeds with plump and uniform size were selected. The surface disinfection steps of the seeds were as follows: plump and uniform tomato seeds were immersed in a 70% ethanol solution for 1 min; the ethanol residue was discarded and the seeds were soaked in a 12% sterile NaClO solution for 10 min; the tomato seeds were rinsed twice with a sterile 2% Na2S2O3 solution; and finally, the tomato seeds were rinsed repeatedly with sterile water (at least 5 times).

[0107] (2) Tomato germination method: Soak tomato seeds in sterile water at room temperature for 10 min; soak in 50℃ warm water for 20 min; soak in 25-30℃ warm water for 4-6 h; place the seeds on gauze and germinate in warm water.

[0108] 2.2.2. The Inducing Effect of Glucan on Bacterial Wilt Disease of Tomato

[0109] Preparation of suspension of *Ralstonia solanacearum*, the pathogen of *Ralstonia solanacearum*, was prepared by spreading *Ralstonia solanacearum* frozen at -80°C onto NA plates (containing 0.005% TTC solution) and activating them at 28°C for 24–48 h. Single colonies were picked and cultured in 5 mL of NB liquid medium at 28°C with shaking at 180 rpm / min until OD600 = 1.0. Fresh *Ralstonia solanacearum* was inoculated into 300 mL of NB liquid medium and cultured at 28°C with shaking for approximately 16 h until OD600 ≈ 0.8. The bacteria were collected by centrifugation at 4000 rpm for 10 min. After discarding the culture medium, the bacteria were washed five times with ddH2O, and the supernatant was discarded. The bacterial cells were resuspended in 10 mL of MgCl2 and adjusted to OD600 = 0.01 (spore concentration approximately 107 cfu / mL) for inoculation.

[0110] To determine the optimal inducing concentration of glucan against bacterial wilt in tomato, glucan was prepared in distilled water at concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mg·L⁻¹. -1 The solution was used for root drenching. When the tomatoes had 4-6 true leaves, the root drenching treatment was performed at a rate of 50 mL per plant. Induction was repeated every 7 days, for a total of 3 inductions. 24 hours after the last induction, approximately 1 / 3 of the tomato plant's root system was cut, and 5 mL of bacterial solution was inoculated near the roots. The water control consisted of tomato plants treated with distilled water and then inoculated with *Ralstonia solanacearum*. The disease index was assessed when the disease was uniform in the water control group. A fungicide control of 100 mg / kg amino oligosaccharide was used. Each treatment was replicated in triplicate, with 30 seedlings per replicate.

[0111] Then, select wild-type tomato plants with similar growth conditions for infection. Place the tomato seedlings in an environment with 28℃, 16 hours of light / 8 hours of darkness, and 70% relative humidity.

[0112] One to three weeks after inoculation, investigate the severity of disease according to the disease grading standards. Grade 0: No symptoms on leaves; Grade 1: 1-20% of leaves wilting; Grade 2: 21-40% of leaves wilting; Grade 3: 41-60% of leaves wilting; Grade 4: 61-80% of leaves wilting; Grade 5: 81% of leaves wilting. -1 00% of the leaves wilted.

[0113] The disease index is calculated using the formula below, and the result is rounded to two decimal places:

[0114]

[0115] In the formula, X is the disease index; Ni is the number of diseased leaves at each level; i is the relative level value; and N is the total number of leaves surveyed.

[0116] The relative prevention and control effect was calculated according to Li Shaobo's method. Calculate as follows, and round the result to two decimal places:

[0117]

[0118] In the formula, P represents the relative prevention and control effect, expressed as a percentage (%); CK represents the disease index of the blank control group; and PT represents the disease index of the drug-treated group.

[0119] 2.3. Statistical Analysis

[0120] GraphPad Prism8 software was used to perform regression analysis on the logarithmic values ​​of drug concentrations and the probability of efficacy, and the EC50 values ​​of each drug were calculated. SPSS (Statistical Procedure for Social Sciences) was used to analyze the significance of differences in disease index among drug treatments.

[0121] 2.4. Results Analysis

[0122] The Inducing Disease Resistance of Tomatoes by Different Concentrations of Dextran

[0123] The effects of different concentrations of glucan on inducing bacterial wilt resistance in tomatoes are shown in Table 1 and... Figure 4 , Figure 5 The results showed that the concentration of glucan in tomato seedlings reached 5 mg·L⁻¹. -1 At that time, resistance developed to some extent, with relative efficacy exceeding 40%. Among them, 50 mg·L... -1 and 20 mg·L -1 Dextran showed the best induction of disease resistance in tomatoes, with relative control efficacies of 65.47% and 62.39% respectively 10 days after inoculation. This was significantly higher than the control fungicide amino oligosaccharide 100 mg·L⁻¹. -1 There was no significant difference in relative efficacy (63.93%). Figure 5 A, 5B), and 20 mg·L -1 The induction period of the treatment was relatively long, and the relative efficacy was still 60.26% 13 days after inoculation, which was higher than the relative efficacy of the fungicide control treatment (47.10%) under the same conditions. Figure 5 (C, 5D). Analysis revealed that the EC50 value of exogenous application of glucan for controlling bacterial wilt in tomatoes was 4.220 mg / kg (Figure 5).

[0124] Table 1. Effect of glucan induction concentration on the induced resistance of tomato to bacterial wilt.

[0125]

[0126] Comparative Example 1

[0127] Table 2. Effects of glucan on powdery mildew in cucurbits

[0128]

[0129] We treated hairy gourd seedlings with 0.5% glucan to prevent powdery mildew in cucurbits. After a second application, powdery mildew occurred in all treatments, indicating that the treatment had no significant effect on preventing powdery mildew.

[0130] in conclusion:

[0131] Experiments show that the glucan of this invention is effective in controlling strawberry anthracnose and tomato bacterial wilt, and its effect on controlling these diseases is better than that of commercially available amino oligosaccharides, representing a significant technological advancement. The glucan of this invention is ineffective against powdery mildew in cucurbits, indicating that it is not effective against all plant diseases. This invention was obtained unexpectedly through extensive experimentation, demonstrating significant efficacy against strawberry anthracnose and tomato bacterial wilt.

[0132] The present invention is not limited to the specific technical solutions described in the above embodiments. All technical solutions formed by equivalent substitutions are within the scope of protection claimed by the present invention.

Claims

1. An application of a glucan, characterized in that... The structural formula of the dextran heptahydrate is as follows: ; The aforementioned glucan is used to prepare agents for treating strawberry anthracnose and tomato bacterial wilt.

2. The application of the heptacapran according to claim 1, characterized in that: The EC50 value for exogenous application of glucan for the control of strawberry anthracnose was 6.072 mg / kg.

3. The application of the heptacapran according to claim 1, characterized in that: The EC50 value for exogenous application of glucan to control bacterial wilt in tomatoes was 4.220 mg / kg.

4. The application of the heptacapran according to claim 1, characterized in that: The method for preparing the dextran heptahydrate includes the following steps: The preparation route of the glucose tetrasaccharide donor is as follows: ； ； ； ； ； Preparation of glucosamine receptors: ； ； Synthesis of the target compound: ； ； ； 。 5. The application of the heptacapran according to claim 1, characterized in that: The method for preparing the dextran heptahydrate includes the following steps: Preparation of glucose tetrasaccharide donors: 1) Dissolve 4.396 g, 5 mmol monosaccharide 1 in 40 ml dichloromethane to obtain solution A; dissolve 1.472 g, 5 mmol monosaccharide 2 in 10 ml dichloromethane to obtain solution B; mix solution A and B to obtain solution C; add 90 μL, 0.5 mmol TMSOTf catalyst to solution C, then add 4A molecular sieve, and react at 25 °C for 3 h. Thin-layer chromatography analysis showed that the reaction was complete. Filter, evaporate the solvent under reduced pressure, separate by column chromatography, and elute with ethyl acetate / cyclohexane (volume ratio 1 / 3) as the eluent. Collect the corresponding fractions to obtain pure disaccharide 3 with a yield of 92%. ； 2) Dissolve 5.056 g (5 mmol) of disaccharide 3 in 50 mL of 80% acetic acid aqueous solution and react at 25 °C for 1-1.5 h. Thin-layer chromatography analysis showed that the reaction was complete. The solvent was removed under reduced pressure, and the mixture was separated by column chromatography. The eluent was ethyl acetate / cyclohexane (volume ratio 1 / 2). The corresponding fractions were collected to obtain pure disaccharide 4. Different acidities affected the yield. The yield was 93% with 70% acetic acid aqueous solution, 95% with 90% acetic acid aqueous solution, and the highest yield (98%) was obtained with 80% acetic acid aqueous solution. ； 3) Dissolve 3.705 g (5 mmol) of monosaccharide 5 in 30 ml of dichloromethane to obtain solution A; dissolve 1.922 g (2.5 mmol) of disaccharide 4 in 20 ml of dichloromethane to obtain solution B. Mix solutions A and B to obtain solution C; add 90 μL (0.5 mmol) of TMSOTf catalyst to solution C, then add 4A molecular sieve. After reacting at 25 °C for 4 h, thin-layer chromatography analysis showed that the reaction was complete. Filter, evaporate the solvent under reduced pressure, separate by column chromatography, and elute with ethyl acetate / cyclohexane at a volume ratio of 1 / 2.

5. Collect the corresponding components to obtain pure tetrasaccharide 6. The yield varies with different reaction concentrations; when the material is at 5 mmol, the yield is 88% when monosaccharide 5 is dissolved in 20 ml of dichloromethane; the yield is 87% when monosaccharide 5 is dissolved in 40 ml of dichloromethane; and the highest yield (91%) is obtained when monosaccharide 5 is dissolved in 30 ml of dichloromethane. ； 4) Dissolve 3.852 g and 2 mmol of tetrasaccharide 6 in 50 ml of 1% hydrochloric acid-methanol solution and react at 25 °C for 2-2.5 h. Thin-layer chromatography analysis showed that the reaction was complete. The solvent was removed under reduced pressure, and the mixture was separated by column chromatography. The eluent was ethyl acetate / cyclohexane (volume ratio 1 / 3). The corresponding fractions were collected to obtain pure tetrasaccharide 7 with a yield of 96%. 5) Dissolve 3.623 g and 2 mmol of tetrasaccharide 7 in 30 mL of dichloromethane, add 0.4 mL and 4 mmol of trichloroacetonitrile, and 0.552 g and 4 mmol of potassium carbonate. Stir at 25 °C for 5 h. Thin-layer chromatography analysis showed that the reaction was complete. Evaporate the solvent under reduced pressure, separate by column chromatography, and use ethyl acetate / cyclohexane at a volume ratio of 1 / 2.5 as the eluent. Collect the corresponding fractions to obtain the tetrasaccharide 8 donor with a yield of 98%. ； ； Preparation of glucosamine receptors: 1) Dissolve 5.056 g, 5 mmol of disaccharide 3 in 50 ml of dichloromethane to obtain solution A; dissolve 3.705 g, 5 mmol of monosaccharide 5 in 10 ml of dichloromethane to obtain solution B; mix solutions A and B to obtain solution C; add 90 μL, 0.5 mmol of TMSOTf catalyst to solution C, then add 4A molecular sieve, and react at 25 °C for 3 h. Thin-layer chromatography analysis showed that the reaction was complete. Filter, evaporate the solvent under reduced pressure, separate by column chromatography, and elute with ethyl acetate / cyclohexane (volume ratio 1 / 3) as the eluent. Collect the corresponding components to obtain pure trisaccharide 9 with a yield of 91%. 2) Dissolve 3.180 g and 2 mmol of trisaccharide 9 in 30 mL of 80% acetic acid aqueous solution and react at 25 °C for 1-1.5 h. Thin-layer chromatography analysis showed that the reaction was complete. The solvent was removed under reduced pressure, and the mixture was separated by column chromatography. The eluent was ethyl acetate / cyclohexane (volume ratio 1 / 3). The corresponding fractions were collected to obtain pure trisaccharide 10 acceptor with a yield of 98%. ； ； Synthesis of the target compound: 1) 2.695 g of 2 mmol glucosamine trisaccharide 10 acceptor, 3.9123 g of 2 mmol glucosamine tetrasaccharide 8 acceptor, and molecular sieve were dissolved in 50 mL of anhydrous dichloromethane and stirred for 1.5 h under nitrogen protection. Then, 72 μL of 0.40 mmol TMSOTf catalyst was added dropwise, and the reaction was carried out at 25 °C for 4.5 h. Thin-layer chromatography analysis showed that the reaction was complete. The mixture was filtered, the solvent was evaporated under reduced pressure, and the mixture was separated by column chromatography. The eluent was ethyl acetate / cyclohexane at a volume ratio of 1 / 1.

8. The corresponding fractions were collected to obtain pure heptaose 11 with a yield of 86%. 2) 6.282 g (2 mmol) of heptaose 11 was dissolved in 35 mL of 1% hydrochloric acid-methanol solution and reacted at 25 °C for 2-2.5 h. Thin-layer chromatography analysis showed that the reaction was complete. The solvent was evaporated under reduced pressure, and the product was separated by column chromatography. The product was eluent with ethyl acetate / cyclohexane at a volume ratio of 1 / 1.8, and the corresponding fractions were collected to obtain pure heptaose 12. Under these conditions, the yield was 95%. Different concentrations of hydrochloric acid affected the yield. The yield was 92% with 0.5% hydrochloric acid-methanol solution and 93% with 1.5% hydrochloric acid-methanol solution. 3) Dissolve 6.054 g and 2 mmol of heptaose 12 in a saturated sodium methoxide solution, stir for 78 h, concentrate, and separate by LH-20 methanol column chromatography with dextran gel to obtain the target product with a yield of 83%. Data of the target product: ESMS m / z 1184{M+NH4}, FW=1166.39; The synthesis reaction formula is as follows: ； ； ； 。

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