Humic acid fertilizer for improving disease resistance of plants

Abstract

The application provides a humic acid fertilizer for improving disease resistance of plants, and belongs to the technical field of agricultural fertilizers.The fertilizer comprises sodium humate, a polysaccharide compound, potassium dihydrogen phosphate, urea, iron sodium ethylenediaminetetraacetate and xanthan gum.The polysaccharide compound is prepared from Capparis sepiara, Ophiopogon japonicus and Atractylodes macrocephala in a mass ratio of 4:3:3, and has a molecular weight range of 30 kDa to 150 kDa.The experimental results show that the humic acid fertilizer can significantly improve the resistance of plants (such as tomato seedlings) to gray mold disease, effectively inhibit the growth of Botrytis cinerea, and promote the recovery and healthy growth of plants after disease.The fertilizer is green and environmentally friendly, and the combination of natural plant polysaccharides and humic acid provides an efficient and safe solution for crop disease control and crop growth.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of agricultural fertilizer, and particularly relates to a humic acid fertilizer for improving disease resistance of plants. BACKGROUND

[0002] Botrytis cinerea, a fungus in the phylum Deuteromycota, is an important plant disease with global distribution. It can infect more than 200 plant species, causing significant damage to high-value crops such as grapes, strawberries, tomatoes, cucumbers, peppers, eggplants, legume vegetables, and ornamental flowers. Botrytis cinerea not only causes persistent infection during the growing season, but also continues to spread during storage and transportation after harvest, leading to water-stained lesions on fruits, leaves, stems, and flowers, and further forming a gray mold layer, ultimately causing tissue rot and significantly reducing the market value and competitiveness of agricultural products.

[0003] Currently, the prevention and control of Botrytis cinerea in agricultural production still mainly relies on chemical fungicides such as benzimidazoles (carbendazim), dicarboximides (iprodione), and methoxy acrylates (azoxystrobin). These chemical pesticides have significant control effects on diseases in the short term, but long-term and large-scale use has brought a series of negative problems. On the one hand, chemical pesticide residues may pollute soil, water, and air, and destroy biodiversity in farmland ecosystems. On the other hand, Botrytis cinerea has developed resistance to many commonly used fungicides, leading to decreased or even ineffective control, increasing the cost and difficulty of control. Therefore, in the face of the dual challenges of increasing disease pressure and ecological environment protection, developing green, safe, and efficient alternative control strategies has become an urgent need for the sustainable development of modern agriculture. SUMMARY

[0004] The present application aims to provide a humic acid fertilizer for improving disease resistance of plants, thereby promoting plant growth and effectively enhancing disease resistance of plants.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] In a first aspect, the present application provides a humic acid fertilizer for improving disease resistance of plants, characterized in that it comprises the following components: sodium humate, polysaccharide complex, potassium dihydrogen phosphate, urea, iron sodium ethylenediaminetetraacetate, and xanthan gum.

[0007] The polysaccharide complex is prepared from Opuntia dillenii Haw, Ophiopogon japonicus, and Atractylodes macrocephala Koidz. in a mass ratio of 4:3:3, and has a molecular weight range of 30 kDa to 150 kDa.

[0008] Preferably, the disease resistance is the ability to resist Botrytis cinerea.

[0009] Preferably, the preparation method of the polysaccharide complex comprises the following steps:

[0010] (1) After the Capparis miclax, Ophiopogon japonicus and Atractylodes macrocephala are crushed into powder, they are mixed according to a mass ratio of 4:3:3;

[0011] (2) Distilled water is added according to a solid-liquid ratio of 1:20 for 2 times of ultrasonic extraction, and the ultrasonic conditions are as follows: temperature 60℃, ultrasonic frequency 40 kHz, ultrasonic power 400 W, pulse mode ultrasonic 2s / pause 1s, and extraction time 40 minutes;

[0012] (3) After the ultrasonic extraction is completed, the extraction liquid is collected by filtration, and the extraction liquid is concentrated under reduced pressure to 1 / 4 of the original volume;

[0013] (4) 3 times of volume of anhydrous ethanol is slowly added, stirred uniformly, and after standing for alcohol precipitation, the crude polysaccharide precipitate is collected by centrifugation;

[0014] (5) The crude polysaccharide is dissolved in distilled water to prepare a crude polysaccharide aqueous solution, and the solution is filtered using a filter membrane;

[0015] (6) The solution is subjected to ultrafiltration using a PES ultrafiltration membrane with a molecular weight cut-off of 150 kDa, and then the permeate is subjected to secondary ultrafiltration using a PES ultrafiltration membrane with a molecular weight cut-off of 30 kDa, and the retentate is collected;

[0016] (7) The retentate is subjected to deproteinization by the Sevag method to obtain a polysaccharide complex solution;

[0017] (8) The polysaccharide complex solution is freeze-dried to obtain a polysaccharide complex.

[0018] Preferably, the humic acid fertilizer is a liquid humic acid fertilizer;

[0019] The combined content of the liquid humic acid fertilizer is 2g of sodium humate, 0.01g to 0.08g of polysaccharide complex, 0.5g of potassium dihydrogen phosphate, 0.5g of urea, 0.1g of iron sodium ethylenediaminetetraacetate and xanthan gum per 100mL of the liquid humic acid fertilizer.

[0020] Preferably, the content of the polysaccharide complex is 0.04g per 100mL of the liquid fertilizer;

[0021] The plant is a tomato seedling.

[0022] Preferably, the preparation method of the liquid humic acid fertilizer comprises the following steps:

[0023] (1) 2g of sodium humate is placed in a beaker, 30mL of deionized water is added, and stirring is performed until complete dissolution to obtain a sodium humate solution;

[0024] (2) Take 0.01-0.08 g of polysaccharide compound D-M2 and place it in a beaker, add 20 mL of deionized water, and stir until completely dissolved to obtain a polysaccharide compound D-M2 solution;

[0025] (3) Take 0.5 g of urea, 0.5 g of potassium dihydrogen phosphate, and 0.05 g of EDTA-FeNa and place them in a beaker, add 20 mL of deionized water, and stir until completely dissolved to obtain an inorganic component solution;

[0026] (4) Take 0.1 g of xanthan gum and place it in a beaker, add 5 mL of deionized water, and stir until a uniform colloid is formed to obtain a stabilizer solution;

[0027] (5) Transfer the humic acid sodium solution, polysaccharide compound D-M2 solution, and inorganic component solution to a 100 mL volumetric flask, then add the stabilizer solution, and dilute to the calibration line with deionized water, and shake well to obtain a mixed solution;

[0028] (6) Pour the mixed solution into a clean beaker, magnetically stir for 10 minutes to ensure uniformity, and finally dispense into light-proof bottles to obtain a humic acid fertilizer containing a polysaccharide compound.

[0029] In a second aspect, the present application provides a polysaccharide compound for use in the preparation of a tomato seedling gray mold protection drug, wherein the polysaccharide compound is prepared by the above preparation method.

[0030] In a third aspect, the present application provides a polysaccharide compound for use in the preparation of a gray mold growth inhibitor, wherein the polysaccharide compound is prepared by the above preparation method.

[0031] In a fourth aspect, the present application provides an antibacterial agent for inhibiting the growth of gray mold, wherein the antibacterial agent is composed of a polysaccharide compound and sterile water, and the polysaccharide compound is prepared by the above preparation method;

[0032] The concentration of the polysaccharide compound in the antibacterial agent is 100-800 μg / mL.

[0033] In a fifth aspect, the present application provides a polysaccharide compound for use in the preparation of a spray for improving the gray mold infection resistance of tomato seedlings, wherein the polysaccharide compound is prepared by the above preparation method.

[0034] In a sixth aspect, the present application provides a spray for improving the gray mold infection resistance of tomato seedlings, wherein the polysaccharide compound is prepared by the above preparation method.

[0035] The concentration of the polysaccharide compound in the spray is 100-800 μg / mL.

[0036] The core of the present application is to innovatively introduce a polysaccharide compound prepared from Capparis zeylanica, Ophiopogon japonicus and Atractylodes macrocephala into humic acid fertilizer, and the polysaccharide compound plays a significant and synergistic role through specific proportions (4:3:3) and accurate molecular weight screening (30 kDa to 150 kDa).

[0037] The beneficial effects of the present application are embodied in particular as follows:

[0038] The polysaccharide compound provided by the present application can significantly inhibit the mycelial growth of Botrytis cinerea, which demonstrates its great potential as a biological bacteriostatic agent.

[0039] Secondly, after the polysaccharide prepared by the present application is prepared into humic acid fertilizer and sprayed, it can effectively inhibit the expansion of the lesion area of gray mold, and at the same time, it can enhance the resistance of tomato seedlings and reduce the proportion of other leaves infected with gray mold.

[0040] In addition, with the comprehensive action of humic acid and active polysaccharide, the fertilizer can not only improve the disease resistance of plants, but also effectively promote the rapid recovery of plants infected with diseases, significantly improve the biomass and fresh weight recovery rate, and ensure the yield and quality of crops.

[0041] Finally, the present application uses natural plant-derived active ingredients to significantly reduce the dependence on traditional chemical pesticides, effectively avoids pesticide residues and environmental pollution, and provides a safe and green solution for the sustainable development of agriculture. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is a detection result graph of the inhibitory effect of different polysaccharide compounds on the mycelial growth of Botrytis cinerea;

[0043] Figure 2 is a detection result graph of the inhibitory effect of polysaccharide compound D-M2 on the mycelial growth of Botrytis cinerea at different concentrations;

[0044] Figure 3 is a comparison result graph of the lesion area of Botrytis cinerea inoculated leaves of tomato seedlings in different treatment groups. DETAILED DESCRIPTION

[0045] The present application will be described in detail below in combination with the drawings and examples.

[0046] Example 1

[0047] (1) The dried Capparis zeylanica, Ophiopogon japonicus and Atractylodes macrocephala purchased from a pharmacy were crushed into powder and passed through a 100-mesh sieve;

[0048] (2) 50 g of Capparis zeylanica powder, 30 g of Ophiopogon japonicus powder and 20 g of Atractylodes macrocephala powder were weighed and mixed into an extraction bottle, distilled water was added according to a solid-liquid ratio of 1:20, and ultrasonic extraction was performed according to the following ultrasonic conditions:

[0049] Temperature: 60°C, ultrasonic frequency: 40 kHz, ultrasonic power: 400 W, pulse mode: ultrasonic 2 s / pause 1 s, extraction time 40 min;

[0050] (3) After the ultrasonic extraction, the residue and the first filtrate were collected, and the residue was placed in the extraction bottle. Distilled water was added according to a solid-liquid ratio of 1:20, and the ultrasonic extraction was performed again under the same conditions;

[0051] (4) The second filtrate was collected, and the first filtrate and the second filtrate were combined to obtain the extraction liquid;

[0052] (5) After the extraction liquid was concentrated to 1 / 4 of the original volume under reduced pressure, 3 times the volume of anhydrous ethanol was slowly added, stirred uniformly, and placed at 4°C for 12 hours. The supernatant was discarded after centrifugation at 4000 rpm / min for 10 minutes, and the crude polysaccharide precipitate was collected;

[0053] (6) The crude polysaccharide was dissolved in distilled water to prepare a 1% crude polysaccharide aqueous solution, and a 0.45 μm filter membrane was used to filter and remove insoluble substances;

[0054] (7) The PES ultrafiltration membrane with a molecular weight cut-off of 150 kDa was used for ultrafiltration at 25°C and 0.2 MPa pressure, and the retained filtrate was recorded as polysaccharide complex A-M1 sample (> 150 kDa);

[0055] (8) The permeate was ultrafiltrated using a PES ultrafiltration membrane with a molecular weight cut-off of 30 kDa at 25°C and 0.2 MPa pressure, and the retained filtrate was recorded as polysaccharide complex A-M2 sample (30-150 kDa);

[0056] (9) The polysaccharide complex A-M1 sample and the polysaccharide complex A-M2 sample were deproteinized using the Sevag method (chloroform:n-butanol=4:1) for 5 times to obtain polysaccharide complex A-M1 solution and polysaccharide complex A-M2 solution;

[0057] (10) The polysaccharide complex A-M1 solution and the polysaccharide complex A-M2 solution were freeze-dried to obtain polysaccharide complex A-M1 and polysaccharide complex A-M2.

[0058] Example 2

[0059] (1) The dried Capparis zeylanica, Ophiopogon japonicus and Atractylodes macrocephala purchased from the pharmacy were crushed into powder and passed through a 100 mesh sieve;

[0060] (2) 30 g of Capparis zeylanica powder, 50 g of Ophiopogon japonicus powder, and 20 g of Atractylodes macrocephala powder were mixed and added to the extraction bottle. Distilled water was added according to a solid-liquid ratio of 1:20, and ultrasonic extraction was performed under the following ultrasonic conditions:

[0061] Temperature: 60°C, ultrasonic frequency: 40 kHz, ultrasonic power: 400 W, pulse mode: ultrasonic 2 s / pause 1 s, extraction time 40 min;

[0062] (3) After the ultrasonic extraction, the residue and the first filtrate were collected, and the residue was placed in the extraction bottle. Distilled water was added according to a solid-liquid ratio of 1:20, and the ultrasonic extraction was performed again under the same conditions;

[0063] (4) The second filtrate was collected, and the first filtrate and the second filtrate were combined to obtain the extraction liquid;

[0064] (5) After the extraction liquid was concentrated to 1 / 4 of the original volume under reduced pressure, 3 times the volume of anhydrous ethanol was slowly added, stirred uniformly, placed at 4°C for 12 hours, centrifuged at 4000 rpm / min for 10 minutes, the supernatant was discarded, and the crude polysaccharide precipitate was collected;

[0065] (6) The crude polysaccharide was dissolved in distilled water to prepare a 1% crude polysaccharide aqueous solution, and a 0.45 μm filter membrane was used to filter and remove insoluble substances;

[0066] (7) The PES ultrafiltration membrane with a molecular weight cut-off of 150 kDa was used for ultrafiltration at 25°C and 0.2 MPa pressure, and the retained filtrate was recorded as polysaccharide complex B-M1 sample (>150 kDa);

[0067] (8) The permeate was ultrafiltrated using a PES ultrafiltration membrane with a molecular weight cut-off of 30 kDa at 25°C and 0.2 MPa pressure, and the retained filtrate was recorded as polysaccharide complex B-M2 sample (30-150 kDa);

[0068] (9) The polysaccharide complex B-M1 sample and the polysaccharide complex B-M2 sample were deproteinized using the Sevag method (chloroform:n-butanol=4:1) to obtain polysaccharide complex B-M1 solution and polysaccharide complex B-M2 solution;

[0069] (10) The polysaccharide complex B-M1 solution and the polysaccharide complex B-M2 solution were freeze-dried to obtain polysaccharide complex B-M1 and polysaccharide complex B-M2.

[0070] Example 3

[0071] (1) The dried Capparis zeylanica, Ophiopogon japonicus and Atractylodes macrocephala purchased from the pharmacy were crushed into powder and passed through a 100-mesh sieve;

[0072] (2) 20 g of Capparis zeylanica powder, 30 g of Ophiopogon japonicus powder, and 50 g of Atractylodes macrocephala powder were mixed and added to the extraction bottle. Distilled water was added according to a solid-liquid ratio of 1:20, and ultrasonic extraction was performed under the following conditions:

[0073] Temperature: 60°C, ultrasonic frequency: 40 kHz, ultrasonic power: 400 W, pulse mode: ultrasonic 2 s / pause 1 s, extraction time 40 min;

[0074] (3) After the ultrasonic extraction, the residue and the first filtrate were collected, the residue was placed in the extraction bottle, and distilled water was added according to a solid-liquid ratio of 1:20. The ultrasonic extraction was performed again under the same conditions;

[0075] (4) The second filtrate was collected, and the first filtrate and the second filtrate were combined to obtain the extraction liquid;

[0076] (5) After the extraction liquid was concentrated to 1 / 4 of the original volume under reduced pressure, 3 times the volume of anhydrous ethanol was slowly added, stirred uniformly, placed at 4°C for 12 hours, centrifuged at 4000 rpm / min for 10 minutes, the supernatant was discarded, and the crude polysaccharide precipitate was collected;

[0077] (6) The crude polysaccharide was dissolved in distilled water to prepare a 1% crude polysaccharide aqueous solution, and a 0.45 μm filter membrane was used to filter and remove insoluble substances;

[0078] (7) The PES ultrafiltration membrane with a molecular weight cut-off of 150 kDa was used for ultrafiltration at 25°C and 0.2 MPa pressure, and the retained liquid was recorded as the polysaccharide complex C-M1 sample (>150 kDa);

[0079] (8) The permeate was ultrafiltrated using a PES ultrafiltration membrane with a molecular weight cut-off of 30 kDa at 25°C and 0.2 MPa pressure, and the retained liquid was recorded as the polysaccharide complex C-M2 sample (30-150 kDa);

[0080] (9) The polysaccharide complex C-M1 sample and the polysaccharide complex C-M2 sample were deproteinized using the Sevag method (chloroform:n-butanol=4:1) to obtain the polysaccharide complex C-M1 solution and the polysaccharide complex C-M2 solution;

[0081] (10) The polysaccharide complex C-M1 solution and the polysaccharide complex C-M2 solution were freeze-dried to obtain the polysaccharide complex C-M1 and the polysaccharide complex C-M2.

[0082] Example 4

[0083] (1) The dried Caper, Ophiopogon and Atractylodes purchased from the pharmacy were crushed into powder and passed through a 100-mesh sieve;

[0084] (2) 40 g of Caper powder, 30 g of Ophiopogon powder, and 30 g of Atractylodes powder were mixed and added to the extraction bottle, distilled water was added according to a solid-liquid ratio of 1:20, and ultrasonic extraction was performed under the following conditions:

[0085] Temperature: 60℃, ultrasonic frequency: 40 kHz, ultrasonic power: 400 W, pulse mode: ultrasonic 2 s / pause 1 s, extraction time 40 min;

[0086] (3) After the ultrasonic extraction, the residue and the first filtrate were collected, the residue was placed in an extraction bottle, distilled water was added according to a solid-liquid ratio of 1:20, and the ultrasonic extraction was performed again under the same conditions;

[0087] (4) The second filtrate was collected, and the first filtrate and the second filtrate were combined to obtain an extraction liquid;

[0088] (5) After the extraction liquid was concentrated to 1 / 4 of the original volume under reduced pressure, 3 times the volume of anhydrous ethanol was slowly added, stirred uniformly, placed at 4℃ for 12 hours, centrifuged at 4000 rpm / min for 10 minutes, the supernatant was discarded, and the crude polysaccharide precipitate was collected;

[0089] (6) The crude polysaccharide was dissolved in distilled water to prepare a 1% crude polysaccharide aqueous solution, and a 0.45 μm filter membrane was used to filter and remove insoluble substances;

[0090] (7) The PES ultrafiltration membrane with a molecular weight cut-off of 150 kDa was used for ultrafiltration at 25℃ and 0.2 MPa pressure, and the retained filtrate was recorded as the polysaccharide complex D-M1 sample (>150 kDa);

[0091] (8) The permeate was subjected to ultrafiltration using a PES ultrafiltration membrane with a molecular weight cut-off of 30 kDa at 25℃ and 0.2 MPa pressure, and the retained filtrate was recorded as the polysaccharide complex D-M2 sample (30-150 kDa);

[0092] (9) The polysaccharide complex C-M1 sample and the polysaccharide complex D-M2 sample were subjected to deproteinization by the Sevag method (chloroform:n-butanol = 4:1) to obtain a polysaccharide complex D-M1 solution and a polysaccharide complex C-M2 solution;

[0093] (10) The polysaccharide complex D-M1 solution and the polysaccharide complex D-M2 solution were freeze-dried to obtain the polysaccharide complex D-M1 and the polysaccharide complex D-M2.

[0094] Example 5

[0095] Detection of the effect of different polysaccharide complexes prepared in the application on the growth of Botrytis cinerea mycelium

[0096] (1) Botrytis cinerea (ATCC58025) was cultured on Potato Dextrose Agar (PDA) and cultured in a 24℃ incubator for 7 days until the mycelium grew;

[0097] (2) The mycelium at the edge of the culture dish was picked up with a sterile inoculation needle and placed in sterile distilled water containing 0.05% (volume fraction) Tween 80. The spores were washed off by stirring, counted using a red blood cell counting plate, and adjusted to a concentration of 1 x 10 6 spores / mL with sterile water;

[0098] (3) The polysaccharide complexes prepared in Examples 1-4 were formulated into PDA medium containing 200 μg / mL (sterile filtered polysaccharide solution was added when cooled to 50°C), and experiments were performed according to the following grouping:

[0099] Control group: PDA medium + 10 μL of spore suspension (inoculated in the center of the medium);

[0100] Treatment group 1: PDA medium containing 200 μg / mL of polysaccharide complex A-M1 + 10 μL of spore suspension;

[0101] Treatment group 2: PDA medium containing 200 μg / mL of polysaccharide complex A-M2 + 10 μL of spore suspension;

[0102] Treatment group 3: PDA medium containing 200 μg / mL of polysaccharide complex B-M1 + 10 μL of spore suspension;

[0103] Treatment group 4: PDA medium containing 200 μg / mL of polysaccharide complex B-M2 + 10 μL of spore suspension;

[0104] Treatment group 5: PDA medium containing 200 μg / mL of polysaccharide complex C-M1 + 10 μL of spore suspension;

[0105] Treatment group 6: PDA medium containing 200 μg / mL of polysaccharide complex C-M2 + 10 μL of spore suspension;

[0106] Treatment group 7: PDA medium containing 200 μg / mL of polysaccharide complex D-M1 + 10 μL of spore suspension;

[0107] Treatment group 8: PDA medium containing 200 μg / mL of polysaccharide complex D-M2 + 10 μL of spore suspension;

[0108] All plates with spore suspension added were placed in an incubator at 24°C for 3 days, after which the diameters of the colonies were counted using a cross method. Three plates were set up for each treatment, and the experiment was repeated three times.

[0109] The inhibition rate was calculated according to the measured colony diameters, and the results are shown in Table 1 and Figure 1 .

[0110] Table 1 Effect of different polysaccharide complexes on the growth of Botrytis cinerea mycelium

[0111]

[0112] From the results of Table 1 and Figure 1 It can be seen that, compared with the control group (colony diameter 52.03 mm), the mycelial growth of Botrytis cinerea was inhibited in different degrees in each treatment group, but the inhibition effect was significantly different. Among them, under the condition of high molecular weight segment (> 150 kDa) (treatment groups 1, 3, 5, 7), the overall antibacterial ability of polysaccharide complex with different ratios was weak, and the inhibition rate was generally lower than 25%, among which the best effect was treatment group 7 (D-M1, ratio 4:3:3), and the inhibition rate was 23.49%.

[0113] In contrast, under the condition of medium molecular weight segment (30-150 kDa) (treatment groups 2, 4, 6, 8), the antibacterial activity of polysaccharide complex was significantly enhanced, especially treatment group 8 (D-M2, ratio 4:3:3) showed the strongest inhibition effect, with a colony diameter of only 29.12 mm and an inhibition rate of 44.03%, which was significantly better than all other treatment groups.

[0114] Under the same molecular weight segment, the antibacterial activity of the four polysaccharide complexes was significantly different, indicating that the ratio of different plant raw materials directly affected the final antibacterial effect. Among them, D ratio (Capparis spinosa: Ophiopogon japonicus: Atractylodes macrocephala = 4:3:3) showed the best antibacterial activity (inhibition rate 44.03%), which was significantly better than other combinations (inhibition rate 22.47%-28.16%).

[0115] The above results fully demonstrate that there is a synergistic optimization relationship between the ratio of Capparis spinosa, Ophiopogon japonicus and Atractylodes macrocephala polysaccharides and molecular weight, and it is not a simple superposition of two independent variables. The inhibition effect of treatment group 8 is much better than that of other groups.

[0116] Example 6

[0117] Detection of the optimal concentration and action concentration range of polysaccharide complex D-M2

[0118] The experimental detection steps are the same as in Example 5, and the groups are as follows:

[0119] Control group: PDA medium + 10 μL spore suspension (inoculated in the center of the medium);

[0120] Treatment group 1: PDA medium containing 100 μg / mL polysaccharide complex D-M2 + 10 μL spore suspension;

[0121] Treatment group 2: PDA medium containing 200 μg / mL polysaccharide complex D-M2 + 10 μL spore suspension;

[0122] Treatment group 3: PDA medium containing 400 μg / mL polysaccharide complex D-M2 + 10 μL spore suspension;

[0123] Treatment group 4: PDA medium containing 800 μg / mL polysaccharide complex D-M2 + 10 μL spore suspension;

[0124] The results of the determination are shown in Table 2. Figure 2

[0125] In Table 2, the colony diameter of the control group was 53.57 ± 2.21 mm, the colony diameter of the treatment group 1 was 47.97 ± 2.30 mm, the colony diameter of the treatment group 2 was 30.35 ± 2.75 mm, the colony diameter of the treatment group 3 was 16.06 ± 1.98 mm, and the colony diameter of the treatment group 4 was 15.32 ± 1.63 mm.

[0126] It can be seen that with the increase of the concentration of polysaccharide complex D-M2, the inhibition effect on the growth of Botrytis cinerea mycelium is obviously enhanced. At a concentration of 100 μg / mL, a certain degree of inhibition effect (10.45%) can be observed, indicating that the polysaccharide complex can exert a certain effect at a lower concentration. At a concentration of 400 μg / mL and 800 μg / mL, the colony diameters are 16.06 mm and 15.32 mm respectively, which are significantly reduced compared with the control group, indicating that the polysaccharide complex has strong antifungal activity at a higher concentration. Further observation shows that the antibacterial effect is slightly improved from 400 μg / mL to 800 μg / mL, showing a nearly saturated inhibition effect.

[0127] Example 7

[0128] Preparation of humic acid fertilizer containing polysaccharide complex I

[0129] For example, 100 mL of liquid fertilizer contains the following formula:

[0130] Sodium humate (self-owned by the company) 2 g, polysaccharide complex D-M2 0.04 g, potassium dihydrogen phosphate (sigma) 0.5 g, urea (sigma) 0.5 g, iron sodium ethylenediaminetetraacetate (EDTA-FeNa, sigma, Cas No.: 149022-26-4) 0.05 g, xanthan gum (sigma) 0.1 g, deionized water 100 mL.

[0131] The preparation method of the humic acid fertilizer is as follows:

[0132] (1) Put 2 g of sodium humate into a beaker, add 30 mL of deionized water, stir until completely dissolved, and obtain a sodium humate solution;

[0133] ​(2) Take 0.04 g of polysaccharide compound D-M2 and place it in a beaker, add 20 mL of deionized water, and stir until completely dissolved to obtain a polysaccharide compound D-M2 solution;

[0134] (3) Place 0.5 g of urea, 0.5 g of potassium dihydrogen phosphate, and 0.05 g of EDTA-FeNa in a beaker, add 20 mL of deionized water, and stir until completely dissolved to obtain an inorganic component solution;

[0135] (4) Place 0.1 g of xanthan gum in a beaker, add 5 mL of deionized water, and stir until a uniform colloid is formed to obtain a stabilizer solution;

[0136] (5) Transfer the humic acid sodium solution, polysaccharide compound D-M2 solution, and inorganic component solution to a 100 mL volumetric flask, then add the stabilizer solution, and dilute to the calibration line with deionized water, and shake well to obtain a mixed solution;

[0137] (6) Pour the mixed solution into a clean beaker, and magnetically stir for 10 minutes to ensure uniformity, and finally dispense into light-proof bottles to obtain a humic acid fertilizer containing polysaccharide compound I.

[0138] Example 8

[0139] Preparation of humic acid fertilizer containing polysaccharide compound II

[0140] Take 100 mL of liquid fertilizer as an example, the formula composition contained is:

[0141] Humic acid sodium 2 g, polysaccharide compound D-M2 0.01 g, potassium dihydrogen phosphate 0.5 g, urea 0.5 g, ethylenediaminetetraacetic acid iron sodium (EDTA-FeNa) 0.05 g, xanthan gum 0.1 g, deionized water 100 mL.

[0142] The preparation method of the humic acid fertilizer is as follows:

[0143] (1) Place 2 g of humic acid sodium in a beaker, add 30 mL of deionized water, and stir until completely dissolved to obtain a humic acid sodium solution;

[0144] (2) Take 0.01 g of polysaccharide compound D-M2 and place it in a beaker, add 20 mL of deionized water, and stir until completely dissolved to obtain a polysaccharide compound D-M2 solution;

[0145] (3) Place 0.5 g of urea, 0.5 g of potassium dihydrogen phosphate, and 0.05 g of EDTA-FeNa in a beaker, add 20 mL of deionized water, and stir until completely dissolved to obtain an inorganic component solution;

[0146] (4) 0.1 g of xanthan gum was placed in a beaker, 5 mL of deionized water was added, and stirred until a uniform colloid was formed to obtain a stabilizer solution;

[0147] (5) The humic acid sodium solution, the polysaccharide complex D-M2 solution and the inorganic component solution were transferred to a 100 mL volumetric flask, then the stabilizer solution was added, and deionized water was added to the calibration line, and shaken well to obtain a mixed solution;

[0148] (6) The mixed solution was poured into a clean beaker and magnetically stirred for 10 minutes to ensure uniformity, and finally divided into light-proof bottles to obtain a humic acid fertilizer II containing a polysaccharide complex.

[0149] Example 9

[0150] Preparation of humic acid fertilizer III containing polysaccharide complex

[0151] For example, 100 mL of liquid fertilizer contains the following formula composition:

[0152] humic acid sodium 2 g, polysaccharide complex D-M2 0.08 g, potassium dihydrogen phosphate 0.5 g, urea 0.5 g, iron sodium ethylenediaminetetraacetate (EDTA-FeNa) 0.05 g, xanthan gum 0.1 g, deionized water 100 mL.

[0153] (1) 2 g of humic acid sodium was placed in a beaker, 30 mL of deionized water was added, and stirred until completely dissolved to obtain a humic acid sodium solution;

[0154] (2) 0.08 g of polysaccharide complex D-M2 was taken and placed in a beaker, 20 mL of deionized water was added, and stirred until completely dissolved to obtain a polysaccharide complex D-M2 solution;

[0155] (3) 0.5 g of urea, 0.5 g of potassium dihydrogen phosphate, and 0.05 g of EDTA-FeNa were placed in a beaker, 20 mL of deionized water was added, and stirred until completely dissolved to obtain an inorganic component solution;

[0156] (4) 0.1 g of xanthan gum was placed in a beaker, 5 mL of deionized water was added, and stirred until a uniform colloid was formed to obtain a stabilizer solution;

[0157] (5) The humic acid sodium solution, the polysaccharide complex D-M2 solution and the inorganic component solution were transferred to a 100 mL volumetric flask, then the stabilizer solution was added, and deionized water was added to the calibration line, and shaken well to obtain a mixed solution;

[0158] (6) The mixed solution was poured into a clean beaker and magnetically stirred for 10 minutes to ensure uniformity, and finally divided into light-proof bottles to obtain a humic acid fertilizer II containing a polysaccharide complex.

[0159] Comparative Example 1

[0160] Humic acid fertilizer prepared using 0.04 g of polysaccharide complex A-M2, except that the polysaccharide complex is different, the rest of the ingredients and preparation method are the same as in Example 7, comparative humic acid fertilizer a.

[0161] Comparative Example 2

[0162] Humic acid fertilizer prepared using 0.04 g of polysaccharide complex B-M2, except that the polysaccharide complex is different, the rest of the ingredients and preparation method are the same as in Example 7, comparative humic acid fertilizer b.

[0163] Comparative Example 3

[0164] Humic acid fertilizer prepared using 0.04 g of polysaccharide complex C-M2, except that the polysaccharide complex is different, the rest of the ingredients and preparation method are the same as in Example 7, comparative humic acid fertilizer c.

[0165] Example 10

[0166] The experimental grouping of this example is as follows:

[0167] Control group: pretreatment spraying of 8 mL of humic acid fertilizer without polysaccharide complex (except that the polysaccharide complex is missing, the rest of the ingredients and preparation method are the same as in Example 7), and subsequent infection of Botrytis cinerea is not performed;

[0168] Infection group: pretreatment spraying of 8 mL of humic acid fertilizer without polysaccharide complex, and subsequent infection of the middle of the second true leaf with 10 μL of spore solution (1 × 10 6 CFU / mL);

[0169] Experimental group: pretreatment spraying of 8 mL of humic acid fertilizer I containing polysaccharide complex, and subsequent infection of the middle of the second true leaf with 10 μL of spore solution (1 × 10 6 CFU / mL);

[0170] Comparative group 1: pretreatment spraying of 8 mL of comparative humic acid fertilizer a, and subsequent infection of the middle of the second true leaf with 10 μL of spore solution (1 × 10 6 CFU / mL);

[0171] Comparative group 2: pretreatment spraying of 8 mL of comparative humic acid fertilizer b, and subsequent infection of the middle of the second true leaf with 10 μL of spore solution (1 × 10 6 CFU / mL);

[0172] Comparative group 3: pretreatment spraying of 8 mL of comparative humic acid fertilizer c, and subsequent infection of the middle of the second true leaf with 10 μL of spore solution (1 × 10 6 CFU / mL);

[0173] 20 seedlings were set in each group, and 3 independent repeats were performed.

[0174] The experimental steps of the present embodiment are as follows:

[0175] 1. Pretreatment stage

[0176] The reagents of each group were evenly sprayed on the front and back of the 4-leaf stage tomato leaf of No. 4 medium vegetable, and placed in a greenhouse with a temperature of 22°C and a humidity of 70% for 24 hours to allow the reagents of each group to be fully absorbed;

[0177] 2. Inoculation and induction of disease

[0178] The second true leaf of each tomato seedling was selected for spore liquid dot inoculation;

[0179] After inoculation, the plants were covered with transparent plastic bags for 24 hours to promote disease development.

[0180] 3. Disease observation

[0181] After 4 days of inoculation, on the 5th day, the typical gray mold spots on the 3rd and 4th true leaves that were not inoculated were observed, and the incidence rate was calculated = number of diseased leaves / total number of leaves x 100%;

[0182] At the same time, the lesion area on the 2nd true leaf was measured;

[0183] 4. Determination of seedling weight

[0184] After 7 days of inoculation, the roots of each group of tomato seedlings were cut off on the 8th day, and the fresh weight of the aboveground part was measured.

[0185] Experimental results:

[0186] The experimental results of the incidence rate are shown in Table 2:

[0187] Table 2 Incidence rate of tomato seedling leaf gray mold

[0188]

[0189] As can be seen from the results in Table 2, compared with the infection group, the incidence rate of the experimental group, comparison group 1 and comparison group 2 and comparison group 3 all showed a significant decrease, and the addition of 30-150 kDa polysaccharide complex of Capparis zeylanica, Ophiopogon japonicus and Atractylodes lancea can play a certain degree of induction of tomato seedling resistance to Botrytis cinerea.

[0190] Further observation can see that, compared with comparison group 1, comparison group 2 and comparison group 3, the incidence rate of the experimental group showed a very significant decrease (the difference was statistically significant), which exceeded the typical linear change, indicating that when the ratio of Capparis zeylanica, Ophiopogon japonicus and Atractylodes lancea is 4:3:3, the synergistic induction effect of the three polysaccharides can be achieved, realizing the synergistic induction of tomato seedling resistance to Botrytis cinerea infection.

[0191] The results of the lesion area on the second true leaf of each treatment group are as follows: Figure 3 As shown.

[0192] exist Figure 3 In the control group, the lesion area was 0 mm, while in the infected group, the lesion area was 33.43 ± 2.91 mm. 2 The lesion area in the experimental group was 8.93 ± 1.12 mm. 2 The lesion area in control group 1 was 18.21 ± 1.97 mm. 2 The lesion area in control group 2 was 15.78 ± 1.64 mm. 2 The lesion area in control group 3 was 23.77 ± 2.08 mm. 2 The results indicate that the polysaccharide-containing complex D-M2 used in the experimental group has excellent direct defense against Botrytis cinerea and can effectively inhibit the pathogen's amplification ability. This effect is significantly better than that of control groups 1, 2 and 3.

[0193] The statistical results of fresh weight and percentage of fresh weight recovery of tomato seedlings in each treatment group 7 days after inoculation with Botrytis cinerea were calculated as follows: (Fresh weight of experimental group - Fresh weight of infected group) / (Fresh weight of control group - Fresh weight of infected group) × 100%

[0194] The results are shown in Table 3.

[0195] Table 3. Fresh weight statistics of tomato seedlings in each treatment group 7 days after inoculation with gray mold.

[0196]

[0197] As shown in Table 3, the infected group experienced stunted plant development and a significant decrease in fresh weight due to Botrytis cinerea infection. The experimental group (using the D-M2 polysaccharide combination, Citrus aurantium: Ophiopogon japonicus: Atractylodes macrocephala = 4:3:3) significantly improved the plant's growth recovery after disease resistance, with a fresh weight recovery percentage of 86.5%, significantly better than all control groups (the difference was statistically significant). Although control groups 1–3 also partially increased biomass, the recovery rate was significantly different from that of the experimental group, indicating that the polysaccharide combination structure has specificity and synergistic effect on disease resistance and growth promotion. These results further verify that the fertilizer prepared by combining the medium molecular weight polysaccharide combination of Citrus aurantium, Ophiopogon japonicus, and Atractylodes macrocephala in a 4:3:3 ratio with humic acid has a significant effect on inducing plant resistance and promoting growth.

Claims

1. A humic acid fertilizer for improving plant disease resistance, characterized in that, The humic acid fertilizer is a liquid humic acid fertilizer; The combined content of the liquid humic acid fertilizer is 2 g of sodium humate, 0.01 g to 0.08 g of polysaccharide compound, 0.5 g of potassium dihydrogen phosphate, 0.5 g of urea, 0.05 g of iron sodium ethylenediaminetetraacetate and 0.1 g of xanthan gum, based on 100 mL of the liquid humic acid fertilizer; The polysaccharide compound has a molecular weight range of 30 kDa to 150 kDa; The disease resistance is the ability to resist gray mold; The preparation method of the polysaccharide compound comprises the following steps: (1) crushing the Capparis micracantha, Ophiopogon japonicus and Atractylodes macrocephala into powders and mixing them according to a mass ratio of 4:3:3; (2) adding distilled water according to a solid-liquid ratio of 1:20 for twice ultrasonic extraction, the ultrasonic conditions being a temperature of 60 DEG C, an ultrasonic frequency of 40 kHz, an ultrasonic power of 400 W, pulse mode ultrasonic 2 s / pause 1 s, and an extraction time of 40 minutes; (3) after the ultrasonic extraction, combining the extraction liquids and reducing the extraction liquids to 1 / 4 of the original volume under reduced pressure; (4) slowly adding 3 times the volume of anhydrous ethanol, stirring uniformly, standing for alcohol precipitation, and centrifugally collecting the crude polysaccharide precipitate; (5) dissolving the crude polysaccharide in distilled water to prepare a crude polysaccharide aqueous solution and filtering using a filter membrane; (6) using a 150 kDa PES ultrafiltration membrane for ultrafiltration, then using a 30 kDa PES ultrafiltration membrane for secondary ultrafiltration of the permeate, and collecting the retentate; (7) deproteinizing the retentate by the Sevag method to obtain a polysaccharide compound solution; (8) freeze-drying the polysaccharide compound solution to obtain the polysaccharide compound.

2. The humic acid fertilizer according to claim 1, characterized in that, The content of the polysaccharide compound is 0.04 g, based on 100 mL of the liquid fertilizer; The plant is a tomato seedling.

3. Use of a polysaccharide complex for the preparation of a protective agent against grey mould of tomato seedlings, characterized in that, The polysaccharide compound is prepared by the preparation method in claim 1.

4. Use of a polysaccharide complex for the preparation of an inhibitor of the growth of Botrytis cinerea, characterized in that, The polysaccharide compound is prepared by the preparation method in claim 1.

5. An antimicrobial agent for inhibiting the growth of Botrytis cinerea, characterized by comprising: The bacteriostatic agent is composed of a polysaccharide compound and sterile water, the polysaccharide compound being prepared by the preparation method in claim 1; The concentration of the polysaccharide compound in the bacteriostatic agent is 100-800 μg / mL.

6. Use of a polysaccharide complex for the preparation of a spray for increasing the resistance to infection by Botrytis cinerea of tomato seedlings, characterized in that, The polysaccharide compound is prepared by the preparation method in claim 1.

7. A spray for improving the infection resistance of tomato seedlings against gray mold, characterized by, The polysaccharide compound is prepared by the preparation method in claim 1. The concentration of the polysaccharide compound in the spray is 100-800 μg / mL.

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