Use of a Herba Eupatorii extract in preparing a product for preventing and treating citrus Huanglongbing disease

Abstract

The present application relates to the technical field related to the prevention and treatment of citrus Huanglongbing, and specifically provides a use of a radix strychnifolia extract in the preparation of a product for preventing and treating citrus Huanglongbing. The present application uses a radix strychnifolia extract to prevent and treat citrus Huanglongbing, which not only has a good prevention and treatment effect, but also has the advantages of low toxicity and high safety for crops.

Description

Technical Field

[0001] The present application relates to the technical field related to the prevention and treatment of citrus Huanglongbing disease, and specifically to the use of a Herba Glehniae extract in the preparation of products for the prevention and treatment of citrus Huanglongbing disease. Background Art

[0002] Citrus Huanglongbing or citrus greening disease is an immune disease caused by phloem-restricted parasites (Candidatus liberibacters spp.). Huanglongbing bacteria belong to the class of α-Proteobacteria and include three tentative species, namely, Asian species (Candidatus Liberibacterasiaticus, Las), African species (Candidatus Liberib acter africanus, Laf) and American species (Candidatus Liberibacter americanus, Lam).

[0003] Citrus Huanglongbing is mainly spread by psyllids and grafting, infecting most Rutaceae plants, such as Citrus: Citrus reticulate Blance, Citrus lemon (L.) Burm.f.; Poncirus: Poncirus trifoliate Rafin; Forttunella: F. crassifolia Swingle, etc. The main symptoms of the disease in the seedling stage are dwarfing of the plant, difficulty in sprouting, and mottled yellowing of the leaves; in the mature stage, it manifests as yellowing of the tips, greening of the fruit skin, and bitterness of the flesh, which seriously affects the yield and commercial value of the fruit.

[0004] So far, there is no pesticide in the field of plant protection that can achieve the same control effect on citrus Huanglongbing, and no pesticide has been registered for the prevention and control of citrus Huanglongbing. Therefore, there is still no effective prevention and control measure for citrus Huanglongbing. Summary of the invention

[0005] In view of this, the present application provides a use of a Herba Coptidis extract in the preparation of a product for preventing and treating citrus Huanglongbing disease.

[0006] In a first aspect, the present application provides a use of a Herba Coptidis extract in the preparation of a product for preventing and treating citrus Huanglongbing disease.

[0007] In some optional embodiments, the extraction method of the Herba Pieris extract is as follows: dried Herba Pieris is crushed to obtain powder;

[0008] placing the powder in a first ethanol aqueous solution, maintaining the solution at (40-60)°C (e.g., 40°C, 45°C, 50°C, 55°C, or 60°C, etc.) for (2-5) hours (e.g., 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours, etc.), filtering to obtain a first filtrate and a filter residue;

[0009] placing the filter residue in a second ethanol aqueous solution, maintaining at (40-60)°C (e.g., 40°C, 45°C, 50°C, 55°C, or 60°C, etc.) for (2-5) hours (e.g., 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours, etc.), filtering to obtain a second filtrate;

[0010] The first filtrate and the second filtrate are combined, the solvent is removed and the mixture is dried to obtain the extract of Radix Glehniae.

[0011] In some optional embodiments, the weight ratio of the powder, the first ethanol aqueous solution and the second ethanol aqueous solution is 1:(5-20):(5-20); for example, 1:10:10, etc.

[0012] In some optional embodiments, the concentration of the first ethanol aqueous solution is (60-90)% (wt); for example, 60% (wt), 65% (wt), 70% (wt), 75% (wt), 80% (wt), 85% (wt) or 90% (wt), etc.

[0013] In some optional embodiments, the concentration of the second ethanol aqueous solution is (60-90)% (wt); for example, 60% (wt), 65% (wt), 70% (wt), 75% (wt), 80% (wt), 85% (wt) or 90% (wt), etc.

[0014] In some optional embodiments, the drying temperature is selected from (50-70)°C; for example, 50°C, 55°C, 60°C, 65°C or 70°C.

[0015] In some optional embodiments, the application method of the extract of Radix Glehniae is selected from at least one of drilling injection, root irrigation and stem and leaf spraying.

[0016] In some specific embodiments, the method of administering the Herba Coptidis extract is selected from perforation injection.

[0017] In some optional embodiments, the method of applying the extract of the Herba Eupatorii is selected from perforation injection, and the amount of the extract of the Herba Eupatorii used in each application is (50-500) mg / plant; for example, 50 mg / plant, 60 mg / plant, 70 mg / plant, 80 mg / plant, 90 mg / plant, 100 mg / plant, 110 mg / plant, 120 mg / plant, 130 mg / plant, 140 mg / plant, 150 mg / plant, 160 mg / plant, 170 mg / plant, 180 mg / plant, 190 mg / plant, 200 mg / plant, 210 mg / plant, 220 mg / plant, 230 mg / plant, 240 mg / plant 0mg / strain, 250mg / strain, 260mg / strain, 270mg / strain, 280mg / strain, 290mg / strain, 300mg / strain, 310mg / strain, 320mg / strain, 330mg / strain, 340mg / strain, 350mg / strain, 360mg / strain, 370mg / strain, 380mg / strain, 390mg / strain, 400mg / strain, 410mg / strain, 420mg / strain, 430mg / strain, 440mg / strain, 450mg / strain, 460mg / strain, 470mg / strain, 480mg / strain, 490mg / strain or 500mg / strain, etc.

[0018] In some specific embodiments, the method of applying the extract of Radix Glechomae var. ...

[0019] In some specific embodiments, the method of applying the extract of Radix Glehniae is selected from root irrigation.

[0020] In some optional embodiments, the method of applying the extract of the Herba Eupatorii is selected from root irrigation, and the amount of the extract of the Herba Eupatorii used in each application is (1-5) g / plant; for example, 1 g / plant, 1.1 g / plant, 1.2 g / plant, 1.3 g / plant, 1.4 g / plant, 1.5 g / plant, 1.6 g / plant, 1.7 g / plant, 1.8 g / plant, 1.9 g / plant, 2 g / plant, 2.1 g / plant, 2.2 g / plant, 2.3 g / plant, 2.4 g / plant, 2.5 g / plant, 2.6g / plant, 2.7g / plant, 2.8g / plant, 2.9g / plant, 3g / plant, 3.1g / plant, 3.2g / plant, 3.3g / plant, 3.4g / plant, 3.5g / plant, 3.6g / plant, 3.7g / plant, 3.8 g / plant, 3.9g / plant, 4g / plant, 4.1g / plant, 4.2g / plant, 4.3g / plant, 4.4g / plant, 4.5g / plant, 4.6g / plant, 4.7g / plant, 4.8g / plant, 4.9g / plant or 5g / plant, etc.

[0021] In some specific embodiments, the application method of the Herba Eupatorii extract is selected from root irrigation, and the amount of the Herba Eupatorii extract used in each application is 2 g / plant.

[0022] In a second aspect, the present application provides a pesticide composition for preventing and controlling citrus Huanglongbing disease, wherein the pesticide composition comprises an extract of Radix Glehniae.

[0023] In some optional embodiments, the pesticide composition further comprises 1,3-dimethylpropyleneurea (CAS No. 7226-23-5, abbreviated as DMPU).

[0024] In some optional embodiments, the weight ratio of the Herba Eupatorii extract to the 1,3-dimethylpropyleneurea is 1:(0.05-0.3); for example, 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.11, 1:0.12, 1:0.13, 1:0.14, 1:0.15, 1:0.16, 1:0.17, 1:0.18, 1:0.19, 1:0.2, 1:0.21, 1:0.22, 1:0.23, 1:0.24, 1:0.25, 1:0.26, 1:0.27, 1:0.28, 1:0.29 or 1:0.3. In some specific embodiments, the weight ratio of the Herba Eupatorii extract to the 1,3-dimethylpropyleneurea is 1:0.2.

[0025] In some optional embodiments, the formulation of the pesticide composition is selected from aqueous solution, soluble liquid, wettable powder or suspension. In some specific embodiments, the formulation of the pesticide composition is selected from aqueous solution.

[0026] This application has the following beneficial effects:

[0027] The present application adopts the extract of Herba Pieris to prevent and control citrus Huanglongbing, which not only has a good prevention and control effect, but also has the advantages of low toxicity and high safety to crops. DETAILED DESCRIPTION

[0028] The present application discloses a use of an extract of Radix Glehniae in the preparation of a product for preventing and treating citrus Huanglongbing. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the purpose. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0029] In order to make the purpose, technical solutions and advantages of the present application clearer, the implementation scheme of the present application will be further described in detail below in conjunction with examples.

[0030] Unless otherwise stated, all reagents used in this application were commercially available.

[0031] Preparation Example 1, Preparation of the extract of Radix Glehniae:

[0032] In this preparation example, the preparation method of the extract of the Herba Eupatoriae lappa is as follows: dried Herba Eupatoriae lappa is crushed to obtain powder; 10 g of the powder is weighed and placed in 100 g of an ethanol aqueous solution with a concentration of 80% (wt), heated to 50°C and kept at 50°C for 3 hours, filtered while hot to obtain a first filtrate and a filter residue; the filter residue is placed in 100 g of an ethanol aqueous solution with a concentration of 80% (wt), heated to 50°C and kept at 50°C for 3 hours, filtered while hot to obtain a second filtrate; the first filtrate and the second filtrate are combined, the solvent is removed, and the mixture is dried at 60°C until constant weight is reached to obtain the Herba Eupatoriae lappa extract.

[0033] Preparation Examples 2-6: Preparation of aqueous solution of Radix Glechomae Extract:

[0034] Table 1, ingredients of the aqueous solution of the extract of the preparation examples 2 to 6:

[0035]

[0036] Among them, the preparation method of the aqueous solution of the extract of the herbaceous wormwood in Preparation Examples 2 to 6 is: weigh the herbaceous wormwood extract, sodium dodecylbenzenesulfonate (CAS No. 25155-30-0), fatty alcohol polyoxyethylene ether (CAS No. 52292-17-8), 1,3-dimethylpropylene urea, N-methylpyrrolidone (CAS No. 872-50-4) and water respectively, and mix them evenly to obtain the aqueous solution of the herbaceous wormwood extract.

[0037] Preparation Example 7, Preparation of Gastrodia elata extract:

[0038] In this preparation example, the preparation method of Gastrodia elata extract is as follows: the dried rhizome of Gastrodia elata is crushed to obtain powder; 10 g of the powder is weighed, placed in 100 g of an ethanol aqueous solution with a concentration of 80% (wt), heated to 50°C and kept at 50°C for 3 hours, filtered while hot to obtain a first filtrate and a filter residue; the filter residue is placed in 100 g of an ethanol aqueous solution with a concentration of 80% (wt), heated to 50°C and kept at 50°C for 3 hours, filtered while hot to obtain a second filtrate; the first filtrate and the second filtrate are combined, the solvent is removed, and the mixture is dried at 60°C until constant weight is reached to obtain a Gastrodia elata extract.

[0039] Preparation Example 8: Preparation of Gastrodia elata extract aqueous solution:

[0040] Table 2, ingredients of the Gastrodia elata extract aqueous solution of Preparation Example 8:

[0041]

[0042] The preparation method of the Gastrodia elata extract aqueous solution in Preparation Example 8 is as follows: Gastrodia elata extract, sodium dodecylbenzene sulfonate, fatty alcohol polyoxyethylene ether and water are weighed respectively, and mixed evenly to obtain the Gastrodia elata extract aqueous solution.

[0043] Test Example 1: Indoor toxicity test of the extract of the herbaceous plant to citrus Huanglongbing bacteria:

[0044] (1-1) Test strains:

[0045] At present, there is no mature system for the pure culture of citrus Huanglongbing bacteria. This is because citrus Huanglongbing bacteria are nutritionally deficient bacteria, lacking five essential nutrients, including histidine, tryptophan, thiamine, phenylalanine and tyrosine, and are difficult to culture on artificial culture media.

[0046] Bradyrhizobium elkanii is closely related to citrus Huanglongbing disease. In this experiment, Bradyrhizobium elkanii was used as an alternative target of citrus Huanglongbing disease to conduct indoor toxicity tests of control agents. Bradyrhizobium elkanii was purchased from the China Industrial Microbiology Collection Center, and the culture medium was CM0009 Rhizobium Medium-1.

[0047] (1-2) Experimental agent: extract of Herba Lycopodii.

[0048] (1-3) Preparation of culture medium:

[0049] LB medium: 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, dilute to 1000 mL with sterile water, adjust pH to 7.0; add 15 g of agar to make LB medium.

[0050] CM0009 Rhizobium medium-1: 1.0 g yeast extract, 10.0 g mannitol, 200 mL soil extract, 15 g agar, dilute to 1000 mL with sterile water, adjust pH = 7.2.

[0051] Soil extract: 50g soil, add 200mL water, boil at 15psi (1psi=6.894KPa) for 1h, filter and add sterile water to make up to 200mL.

[0052] (1-4) Preparation of Bradyrhizobium elsdenii suspension:

[0053] Use an inoculation loop to pick up fresh colonies and inoculate them into 50 mL of CM0009 rhizobium medium-1. Incubate in a shaker at 28°C and 180 rpm for more than 6 h. Measure the absorbance at 600 nm every 1 h until the bacterial density OD reaches 0. 600nm=1.

[0054] (1-5) Indoor toxicity assay:

[0055] Use a pipette to draw 1 mL of Bradyrhizobium elsdenii suspension and add it to a glass bottle containing 48 mL of CM0009 Rhizobium medium-1 to prepare a bacterial culture medium;

[0056] Dilute the test drugs into concentration gradients with sterile water;

[0057] Use a pipette to draw 1 mL of the diluted test agent and add it to the bacterial culture medium to obtain a series of culture solutions with gradient concentrations of the test agent; use sterile water as a blank control; set up 3 replicates for each treatment;

[0058] The culture was shaken at 28°C and 180 rpm for 12 h, and the OD was measured using a UV spectrophotometer. 600nm The toxicity regression equation was established, and the correlation coefficient r and the half-maximal effect concentration EC50 were calculated. The results of the indoor toxicity test are shown in Table 3.

[0059] Table 3, indoor toxicity test results of the extract of the Chinese hollyhock extract against citrus Huanglongbing bacteria:

[0060]

[0061] As can be seen from Table 3, the extract of Radix Glehniae has a good inhibitory effect on Bradyrhizobium elsdenii, with an EC50 of 12.4556 mg / L.

[0062] Test Example 2: Field control test of citrus Huanglongbing based on the method of drilling and injection:

[0063] In this test case, a citrus orchard with consistent Huanglongbing disease severity and uniform growth was selected. Through real-time fluorescence quantitative PCR (qRT-PCR) detection, 50 positive plants with similar growth severity and bacterial load were selected as test subjects.

[0064] (2-1) Test agents: the aqueous solution of the extract of the Herba Glechomae var. erythrorhizon of Preparation Example 2 (without DMPU), the aqueous solution of the extract of the Herba Glechomae var. erythrorhizon of Preparation Example 3 (the weight ratio of the extract of the Herba Glechomae var. erythrorhizon to DMPU is 1:0.1), the aqueous solution of the extract of the Herba Glechomae var. erythrorhizon of Preparation Example 4 (the weight ratio of the extract of the Herba Glechomae var. erythrorhizon to DMPU is 1:0.2), the aqueous solution of the extract of the Herba Glechomae var. erythrorhizon of Preparation Example 5 (the weight ratio of the extract of the Herba Glechomae var. erythrorhizon to DMPU is 1:0.3), the aqueous solution of the extract of the Herba Glechomae var. erythrorhizon of Preparation Example 6 (containing NMP), the aqueous solution of the extract of the Herba Glechomae var. erythrorhizon of Preparation Example 8, oxytetracycline, carvacrol, and 20% flumorph wettable powder (purchased from Sinochem Agrochemical Co., Ltd.).

[0065] (2-2) Detection of the antioxidant activities of the extracts of Radix Gastrodiae, Rhizoma Gastrodiae, carvacrol and flumorph:

[0066] The antioxidant activities of the extracts of Radix Gastrodiae, Radix Gastrodiae, carvacrol and flumorph were detected by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The specific detection method includes the following steps:

[0067] Accurately weigh DPPH (19.716 mg), dissolve it in anhydrous ethanol and dilute it to a 500 mL volumetric flask. Shake well to obtain 0.1 mmol•L -1 DPPH ethanol solution; store at (0-4)℃ away from light, use immediately after preparation, and is effective within 4 hours.

[0068] The antioxidant was accurately weighed and anhydrous ethanol was used as a solvent to prepare a sample test solution.

[0069] Prepare three EP tubes and record them as tube A, tube B, and tube C. First, add reagents to tube A, tube B, and tube C according to Table 4. Pipette the sample test solution in a gradient manner and add it to tube A, then add distilled water to make it up to 2 mL. Make a parallel for each sample test solution. Then, place it in a light-proof water bath at 37°C for 30 minutes. After the reaction is completed, measure the absorbance (A) of the solution in tube A, tube B, and tube C at 517 nm and record them as , , .

[0070] Table 4. Material-liquid ratio in tube A, tube B and tube C in the detection of antioxidant activity:

[0071]

[0072] First, calculate the DPPH free radical scavenging rate of the sample test solution. The calculation formula of DPPH free radical scavenging rate is: ; Then, the sample concentrations were used as the horizontal axis and the DPPH free radical scavenging rate was used as the vertical axis to make a regression equation curve and calculate the half scavenging concentration (IC50 value). The larger the IC50 value, the smaller the antioxidant activity.

[0073] The test results of antioxidant activity are shown in Table 5.

[0074] Table 5. Antioxidant activity test results:

[0075]

[0076] As can be seen from Table 5, the antioxidant activities of Gastrodia elata extract, carvacrol and flumorph were significantly higher than those of Gastrodia elata extract.

[0077] (2-3) Methods of field control experiment on citrus Huanglongbing:

[0078] In this test example, there are 7 test groups and 1 blank control group, and 5 positive plants are randomly selected from each group. The positive plants in each group are injected with pesticides by punching.

[0079] The test group 1 was treated with the aqueous solution of the extract of the preparation example 2 (without DMPU), and the preparation method was as follows: 1 g of the aqueous solution of the extract of the preparation example 2 was weighed and diluted to 500 mL with distilled water. The injection volume of each positive plant in the test group 1 was 500 ml / time, and the injection amount was 0.1 g (calculated as the extract of ...

[0080] The test group 2 was treated with the aqueous solution of the extract of the Herba Coptidis in Preparation Example 3 (the weight ratio of the Herba Coptidis in Preparation Example 3 to DMPU was 1:0.1), and the preparation method was as follows: 1 g of the aqueous solution of the Herba Coptidis in Preparation Example 3 was weighed and diluted to 500 mL with distilled water. The injection volume of each positive plant in the test group 2 was 500 ml / time, and the injection amount was 0.1 g (calculated as the Herba Coptidis in Preparation Example 3) / time.

[0081] The test group 3 was treated with the aqueous solution of the extract of the preparation example 4 (the weight ratio of the extract ...

[0082] The test group 4 was treated with the aqueous solution of the extract of the preparation example 5 (the weight ratio of the extract ...

[0083] Test group 5 was treated with the aqueous solution of the extract of the herbaceous vine of preparation example 6 (containing NMP), and the preparation method was as follows: weigh 1 g of the aqueous solution of the extract of the herbaceous vine of preparation example 6 and dilute it to 500 mL with distilled water. The injection volume of each positive plant in test group 5 was 500 ml / time, and the injection amount was 0.1 g (calculated as the extract of the herbaceous vine) / time.

[0084] Oxytetracycline was applied to test group 6. The preparation method was as follows: 0.1 g of oxytetracycline was weighed, dissolved in methanol, and then diluted to 500 ml with distilled water. The injection volume of each positive plant in test group 6 was 500 ml / time, and the injection amount was 0.1 g (in terms of oxytetracycline) / time.

[0085] The test group 7 was administered with the aqueous solution of the Gastrodia elata extract of Preparation Example 8, and the preparation method thereof was as follows: 1 g of the aqueous solution of the Gastrodia elata extract of Preparation Example 8 was weighed and diluted to 500 mL with distilled water. The injection volume of each positive plant in the test group 7 was 500 ml / time, and the injection amount was 0.1 g (calculated as the Gastrodia elata extract) / time.

[0086] Test group 8 applied carvacrol, and its preparation method was as follows: weigh 0.1g of carvacrol, dissolve it in methanol, and then dilute it to 500ml with distilled water. The injection volume of each positive plant in test group 5 was 500ml / time, and the injection amount was 0.1g (calculated as carvacrol)mg / time.

[0087] Test group 9 was treated with 20% flumorph wettable powder, the preparation method of which was as follows: 0.5 g of 20% flumorph wettable powder was diluted to 500 mL with distilled water. The injection volume of each positive plant in test group 9 was 500 ml / time, and the injection amount was 0.1 g (calculated as flumorph) / time.

[0088] The blank control group was administered with distilled water, and the injection volume for each positive plant in the blank control group was 500 ml / time.

[0089] Table 6, Grouping of positive plants in Test Example 2:

[0090]

[0091] The specific process of drilling and injecting medicine is as follows: use a 4mm drill bit to drill a medicine injection hole 20 cm below the first branch of the positive plant, with a depth of (2-3) cm. Hang the infusion bag containing the medicine on the branch, empty the air from the infusion tube and place it in the injection hole until no water drips, and adjust the dripping speed of the medicine solution to about 1 drop / min.

[0092] Re-apply the pesticide every 20 days. Before each application, collect 5 suspected diseased leaves from the five directions of east, west, south, north and top of the positive plants. Mix the collected leaves from the same positive plants in self-sealing bags and store them in a 4°C refrigerator to facilitate the subsequent determination of the relative expression level of citrus Huanglongbing disease.

[0093] (2-4) Extraction of deoxyribonucleic acid (DNA):

[0094] Step S1, randomly select 5-10 leaves from each positive plant, take the mesophyll near the main vein of the leaf (with the main vein), cut it into pieces, add liquid nitrogen and grind it into powder, and use an EP tube (specification of 2 mL) to take an appropriate amount of powder sample.

[0095] Step S2, add 800 μL of CTAB buffer to the EP tube containing the powdered sample and vortex mix, place in a 60°C water bath for 45 min, and oscillate to mix every 15 min; then add 800 μL of a mixed solution prepared with phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:1, mix by inverting, let stand for 5 min, and then centrifuge at 12000 rpm for 10 min.

[0096] Step S3: aspirate 600 μL of the supernatant and transfer it to a new EP tube (2 mL), add 600 μL of a mixed solution of chloroform and isoamyl alcohol in a volume ratio of 24:1, mix by inverting, let stand for 5 min, and then centrifuge at 12000 rpm for 10 min.

[0097] Step S4: aspirate 500 μL of the supernatant and transfer it to a new EP tube (1.5 mL in size), add 500 μL of isopropanol, gently invert to mix, precipitate at -20°C for at least 30 min, and centrifuge at 12,000 rpm for 10 min.

[0098] Step S5: discard the supernatant, add 1 mL of 75% ethanol aqueous solution, use a 1000 μL range pipette to blow repeatedly to remove the DNA flakes attached to the EP tube wall, and centrifuge quickly for 15 seconds. Repeat this step once.

[0099] Step S6, discard the supernatant, centrifuge at 12000rpm for 3min, and aspirate the remaining liquid in the EP tube; open the lid and air-dry for 10min. After the EP tube is completely dry, add 50μL of double deionized water to dissolve it to obtain a DNA solution, and store it at -20℃.

[0100] (2-5) Based on the qRT-PCR method, the relative bacterial content of positive plants was determined, and the control effect of citrus Huanglongbing in each test group was calculated:

[0101] The DNA solution of each sample was subjected to 3 repeated qRT-PCR amplification reactions and tested. In the qRT-PCR test, the sequence information of the target gene primers and the internal reference gene primers is shown in Table 7.

[0102] Table 7. Sequence information of target gene primers and internal reference gene primers in qRT-PCR detection:

[0103]

[0104] The composition of each real-time fluorescence quantitative PCR reaction system is shown in Table 6.

[0105] Table 8. Composition of the reaction system in qRT-PCR detection:

[0106]

[0107] The conditions of real-time fluorescence quantitative PCR amplification reaction were: 95℃ pre-denaturation for 2 min; (95℃ extension for 10 s, 60℃ annealing for 30 s) 40 cycles. After the reaction, the standard curve, amplification curve, concentration and sample Ct value were generated.

[0108] The ΔΔCt method was used to perform relative quantitative calculation of the relative bacterial content of positive plants in each test group. The calculation method is as follows:

[0109] ;

[0110] ;

[0111] ;

[0112] .

[0113] in, The blank control group was tested by real-time fluorescence quantitative PCR three times. Therefore, each positive plant in the test group and the blank control group corresponds to a ΔΔCt value.

[0114] .

[0115] The relative bacterial content of the positive plants in the experimental group was normalized with the blank control group as the benchmark, and the relative bacterial content of the positive plants in the blank control group was defined as 1; the control effect of citrus Huanglongbing in the experimental group was calculated.

[0116] .

[0117] Among them, the test results of the prevention and control effect of citrus Huanglongbing in Test Example 2 are shown in Table 9.

[0118] Table 9, control effect of citrus Huanglongbing in test example 2:

[0119]

[0120] As can be seen from Table 9, for the prevention and control effect of citrus Huanglongbing, the extract of Gastrodia elata is significantly better than oxytetracycline, Gastrodia elata extract, carvacrol and flumorph.

[0121] By comparing test groups 1, 3 and 5, it can be seen that DMPU and NMP are both used as penetrants, among which DMPU greatly improves the control effect of the extract of the Herba Lycopodii on citrus Huanglongbing. It can be seen from the experimental data that the weight ratio of the extract of the Herba Lycopodii and DMPU is 1:0.2. At this time, the dosage of DMPU is small, but the synergistic effect is very good; however, NMP not only did not further improve the control effect of the extract of the Herba Lycopodii on citrus Huanglongbing, but also reduced the control effect of the extract of the Herba Lycopodii on citrus Huanglongbing.

[0122] Test Example 3: Field control test of citrus Huanglongbing based on root irrigation:

[0123] In this experiment, the test site was conducted in Baimangying citrus orchard, Jianghua County, Yongzhou City, Hunan Province from 2023 to 2024, and the citrus variety was Eyuan No. 38 (Jelly Orange), with a tree age of 5 years.

[0124] The citrus trees used in this experiment were all tested and retested through endpoint PCR to confirm that they were positive plants; these positive plants were marked for subsequent pesticide application tests, sample collection, and appearance grading investigations.

[0125] (3-1) Test agent: aqueous solution of the extract of the Herba Lysimachiae of Preparation Example 4 (the weight ratio of the Herba Lysimachiae extract to DMPU is 1:0.2).

[0126] (3-2) Methods of field control experiment on citrus Huanglongbing:

[0127] The positive plants were randomly divided into two groups, including one test group and one blank control group, with two positive plants in each group. The positive plants in each group were irrigated with pesticides.

[0128] The test group was applied with the aqueous solution of the extract of the Herba Coptidis (containing DMPU) of Preparation Example 3, and the preparation method thereof was as follows: the aqueous solution of the extract of the Herba Coptidis (containing DMPU) of Preparation Example 3 was prepared with water at a dilution multiple of 500 times. The root irrigation amount of each positive plant in the test group was 10 kg / time, and the application amount was 20 g (calculated as the aqueous solution of the Herba Coptidis extract) / time or 2 g (calculated as the Herba Coptidis extract) / time.

[0129] The blank control group was irrigated with tap water. The root irrigation amount of each positive plant in the blank control group was 10 kg (in terms of water) per time.

[0130] Table 10, Grouping of positive plants in Test Example 3:

[0131]

[0132] The number of root irrigation is 4 times, the first root irrigation is on October 23, 2023, the second root irrigation is on November 13, 2023, the third root irrigation is on December 4, 2023, and the fourth root irrigation is on December 25, 2023. The number of sampling is 5 times, the first sampling is on October 23, 2023, the second sampling is on November 13, 2023, the third sampling is on December 4, 2023, the fourth sampling is on December 25, 2023, and the fifth sampling is on March 19, 2024; and when the time of root irrigation and sampling is on the same day, sampling is done first and then root irrigation. The sampling method is to collect fully expanded new leaves of the current year distributed in the five directions of east, south, west, north and center of each positive plant, and collect 5-10 leaves in each direction. In each sampling, the collected leaves of the same positive plant are mixed in an ice box and brought back to the laboratory and stored in a -80℃ ultra-low temperature refrigerator for the subsequent determination of the relative expression of citrus Huanglongbing bacteria and the determination of physiological and biochemical indicators.

[0133] (3-3) DNA extraction: Same as step (2-4) in Experimental Example 2.

[0134] (3-4) End-point PCR (polymerase chain reaction) detection:

[0135] The DNA solution of each sample was subjected to two end-point PCR tests, wherein the sequence information of the target gene primers is shown in Table 11.

[0136] Table 11. Sequence information of target gene primers in endpoint PCR detection:

[0137]

[0138] Among them, the composition of the reaction system for each end-point PCR detection is shown in Table 12.

[0139] Table 12. Composition of the reaction system in the end-point PCR detection:

[0140]

[0141] The conditions of the end-point PCR amplification reaction were: 96℃ pre-denaturation for 5 min; (95℃, 45 s; 55℃, 30 s; 72℃, 60 s) 35 cycles; 72℃ extension for 7 min. Afterwards, the amplified product was tested by DNA agarose gel electrophoresis; if an obvious 1167 bp band appeared, it indicated that the sample was a positive plant for citrus Huanglongbing.

[0142] (3-5) Determine the relative bacterial content of positive plants based on the qRT-PCR method: Same as step (2-5) in Experimental Example 2.

[0143] Among them, the detection results of the relative bacterial content of the positive plants in Test Example 3 are shown in Table 13.

[0144] Table 13, Test results of relative bacterial content of positive plants in Test Example 3:

[0145]

[0146] As can be seen from Table 13, the relative expression of CLas measured in the third sampling (after the second root irrigation and before the third root irrigation), the fourth sampling (after the third root irrigation and before the fourth root irrigation) and the fifth sampling (after the fourth root irrigation and before the fifth root irrigation) of positive plants No. 1 and No. 2 in the test group was less than 1. This shows that root irrigation of positive plants with the extract of Ganfengchai can effectively inhibit the development of citrus Huanglongbing and reduce the amount of bacteria in positive plants.

[0147] (3-6) Survey on the severity of citrus Huanglongbing disease:

[0148] Before each application of pesticides and before the last sampling, the plant appearance disease classification survey and statistics were conducted. The grading standards for citrus Huanglongbing disease are shown in Table 14.

[0149] Table 14. Citrus greening disease classification standards:

[0150]

[0151] Among them, the survey results of the disease level of citrus Huanglongbing are shown in Table 15.

[0152] Table 15. Survey results of citrus Huanglongbing disease levels:

[0153]

[0154] As can be seen from Table 15, the initial disease level of each positive plant in the test group and the blank control group was 4; among them, the disease level of positive plant No. 1 in the test group was reduced from 4 to 3 after 3 root drenching with the extract of Ganfengchai, and further reduced to 2 after 4 root drenching; the disease level of positive plant No. 2 was reduced from 4 to 3 after 2 root drenching with the extract of Ganfengchai, and then remained at 3. It can be seen that the extract of Ganfengchai has a good control effect on citrus Huanglongbing and effectively reduces the disease level of positive plants.

[0155] (3-7) Determination of chlorophyll content in leaves:

[0156] The method for determining the chlorophyll content in leaves is the spectrophotometer method, and the solvent used to extract chlorophyll is an ethanol aqueous solution with a volume concentration of 95%.

[0157] The absorbance of the test sample was measured at wavelengths of 663nm, 646nm, and 470nm, and recorded as A 663nm , A 646nm , A 470nm Each test sample was measured three times at each wavelength.

[0158] According to A 663nm , A 646nm , A 470nm , calculate the content of chlorophyll a, chlorophyll b and carotenoids; the calculation formula is as follows:

[0159] ;

[0160] ;

[0161] .

[0162] Among them, Ca represents the chlorophyll a content, in mg / g;

[0163] Cb represents the chlorophyll b content, in mg / g;

[0164] Cx represents the carotenoid content in mg / g.

[0165] Among them, the determination results of chlorophyll a content in positive plants are shown in Table 16; the determination results of chlorophyll b content are shown in Table 17; and the determination results of carotenoid content are shown in Table 18.

[0166] Table 16. Results of determination of chlorophyll a content in positive plants:

[0167]

[0168] Table 17, the results of determination of chlorophyll b content in positive plants:

[0169]

[0170] Table 18, determination results of carotenoid content in positive plants:

[0171]

[0172] From Tables 16 to 18, it can be seen that in the positive plants No. 1 and No. 2 in the test group, the chlorophyll a content, chlorophyll b content, and carotenoid content measured in each of the 1st to 5th samplings all showed a trend of gradual increase. This shows that the extract of the Herba Eupatorii can promote the stable increase of chlorophyll content in the positive plants.

[0173] (3-8) Determination of L-phenylalanine ammonia-lyase (PAL):

[0174] Use enzyme activity detection kits (purchased from Beijing Solebow Technology Co., Ltd., catalog numbers BC0215 and BC0175), and strictly follow the steps described in the instructions; then, use a multi-function microplate reader (Multi-function microplate reader, manufactured by Tecan, model spark enhanced) to measure the OD value and calculate the enzyme activity of PAL.

[0175] Due to the differences in the initial bacterial load of different positive plants and the influence of seasonal changes, the increase ratio of PAL enzyme activity of the nth (n=2, 3, 4 or 5) sampling was used to compare the effects of each group on PAL enzyme activity. The calculation formula for the increase ratio of PAL enzyme activity of the nth sampling is as follows:

[0176] .

[0177] Among them, the detection results of enzyme activity and enhancement ratio are shown in Table 19.

[0178] Table 19. Test results of enzyme activity and enhancement ratio in positive plants:

[0179]

[0180] As can be seen from Table 19, the enzyme activity values ​​of positive plants No. 3 and No. 4 in the blank control group showed a trend of "first decreasing and then increasing", and the enzyme activity reached the lowest value in the fourth sampling (after the third root irrigation and before the fourth root irrigation). The enzyme activity values ​​of positive plants No. 1 and No. 2 in the experimental group showed the same change pattern as the positive control group.

[0181] In summary, the extract of the herbaceous twig in the experimental group can reduce the PAL activity and disease level of the positive plants with higher disease levels, as well as the PAL enhancement ratio.

[0182] The above is a detailed introduction to the use of a Herba Coptidis extract provided by the present application in the preparation of products for preventing and treating citrus Huanglongbing. This article uses specific examples to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. Use of a radix strychnifolia extract in the preparation of a product for preventing and treating citrus Huanglongbing; The extraction method of the extract of the Chinese yew is as follows: dried Chinese yew is crushed to obtain powder; placing the powder in a first ethanol aqueous solution, maintaining it at (40-60)° C. for (2-5) hours, filtering to obtain a first filtrate and a filter residue; placing the filter residue in a second ethanol aqueous solution, maintaining it at (40-60)° C. for (2-5) hours, filtering it, and obtaining a second filtrate; The first filtrate and the second filtrate are combined, the solvent is removed and the mixture is dried to obtain the extract of Radix Glehniae; The weight ratio of the powder, the first ethanol aqueous solution and the second ethanol aqueous solution is 1:(5-20):(5-20).

2. A pesticide composition for preventing and treating citrus Huanglongbing, characterized in that: The pesticide composition comprises an extract of Radix Glehniae; The extraction method of the extract of the Chinese yew is as follows: dried Chinese yew is crushed to obtain powder; placing the powder in a first ethanol aqueous solution, maintaining it at (40-60)° C. for (2-5) hours, filtering to obtain a first filtrate and a filter residue; placing the filter residue in a second ethanol aqueous solution, maintaining it at (40-60)° C. for (2-5) hours, filtering it, and obtaining a second filtrate; The first filtrate and the second filtrate are combined, the solvent is removed and the mixture is dried to obtain the extract of Radix Glehniae; The weight ratio of the powder, the first ethanol aqueous solution and the second ethanol aqueous solution is 1:(5-20):(5-20).

3. The pesticide composition according to claim 2, characterized in that The pesticide composition further comprises 1,3-dimethylpropyleneurea.

4. The pesticide composition according to claim 3, characterized in that The weight ratio of the Herba Lysimachiae extract to the 1,3-dimethylpropyleneurea is 1:(0.05-0.3).

5. The pesticide composition according to any one of claims 2 to 4, characterized in that The pesticide composition is selected from a soluble liquid, a wettable powder or a suspension.

6. The pesticide composition according to any one of claims 2 to 4, characterized in that The pesticide composition is selected from aqueous solutions.

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