Leaf fertilizer for improving disease resistance of panax vietnamensis

Abstract

The present application belongs to the technical field of fertilizer, and specifically discloses a foliar fertilizer for improving the disease resistance effect of Vietnamese ginseng, which is composed of 0.1-0.5% potassium dihydrogen phosphate, 0.1-0.2% boric acid, 0.3-0.7% urea, 0.005-0.02% chitosan, 0.0001% salicylic acid and 0.05% Tween-20, and the rest is water, according to the weight percentage. The foliar fertilizer prepared by the present application has a remarkable disease control effect on Vietnamese ginseng, and has a positive control effect on the infection of Fusarium oxysporum, Alternaria panax and Botryosphaeria dothidea. After using the foliar fertilizer of the present application, the growth of the pathogen is significantly inhibited from the phenotype, and the disease onset time of Vietnamese ginseng is significantly later than that of the control. The resistance gene expression of Vietnamese ginseng is significantly improved through testing.

Description

Technical Field

[0001] This invention belongs to the field of fertilizer technology, specifically relating to a foliar fertilizer that enhances the disease resistance of Vietnamese ginseng. Background Technology

[0002] Vietnamese ginseng (Vietnamese ginseng), also known as "Yuan Duong Nam Chi," is a plant belonging to the genus Panax in the family Araliaceae. First recorded in the *Yuan Duong County Gazetteer* (1990), it was used as an ingredient in cold medicine formulations. The *Vietnamese Pharmacopoeia* states that this variety "greatly replenishes vital energy and benefits the lungs," and can treat "physical weakness and near collapse, lung deficiency with cough and asthma, and sore throat." Modern pharmacological studies have shown that Vietnamese ginseng contains a wide range of bioactive components, exhibiting certain medicinal effects in anti-inflammatory, anti-cancer, anti-myocardial ischemia, and hypoglycemic properties.

[0003] Various diseases frequently occur during ginseng cultivation in Vietnam, affecting yields. Currently, chemical control remains the primary method, but this approach carries risks such as pesticide residues, pathogen resistance, environmental pollution, and ecosystem damage. Therefore, finding a green control method is essential for managing ginseng diseases in Vietnam. Summary of the Invention

[0004] The main objective of this invention is to provide a foliar fertilizer that enhances the disease resistance of Vietnamese ginseng, thereby addressing the problems existing in the background art. Specifically, this invention provides the following technical solution:

[0005] The application of a foliar fertilizer in enhancing the disease resistance of Vietnamese ginseng, wherein the foliar fertilizer, by weight percentage, consists of 0.1-0.5% potassium dihydrogen phosphate, 0.1-0.2% boric acid, 0.3-0.7% urea, 0.005-0.02% chitosan, 0.0001% salicylic acid, and 0.05% Tween-20, with the balance being water.

[0006] Furthermore, the foliar fertilizer, by weight percentage, consists of 0.3% potassium dihydrogen phosphate, 0.1% boric acid, 0.5% urea, 0.01% chitosan, 0.0001% salicylic acid, and 0.05% Tween-20, with the balance being water.

[0007] Furthermore, the application is to enhance the resistance of Vietnamese ginseng to diseases caused by Fusarium oxysporum, Alternaria ginseng, or Staphylococcus aureus.

[0008] Furthermore, the application is to enhance the resistance of Vietnamese ginseng to root rot, black spot, or gray mold.

[0009] Furthermore, the preparation method of the foliar fertilizer is as follows:

[0010] S1: Weigh out the chitosan powder and dissolve it in glacial acetic acid;

[0011] S2: Weigh out salicylic acid powder and dissolve it in anhydrous ethanol;

[0012] S3: Dissolve the appropriate amounts of chitosan and salicylic acid, as well as potassium dihydrogen phosphate, boric acid, urea and Tween-20 in water to prepare a foliar fertilizer of the required concentration.

[0013] Beneficial effects of this invention: The foliar fertilizer prepared by this invention has a significant effect on the prevention and control of diseases in Vietnamese ginseng. It has a positive control effect on Fusarium oxysporum, Alternaria ginseng, and Botrytis cinerea. Phenotypically, after using the foliar fertilizer of this invention, the growth of pathogens was significantly inhibited, and the onset of disease in Vietnamese ginseng was significantly later than that in the control group. By testing the expression level of disease resistance genes in Vietnamese ginseng, it was found that the expression level of resistance genes in Vietnamese ginseng was significantly increased after using this foliar fertilizer. Attached Figure Description

[0014] Figure 1 Phenotypic images of Vietnamese ginseng treated with foliar fertilizer after inoculation with Fusarium oxysporum, Alternaria ginseng, and Botrytis cinerea for 0 days.

[0015] Figure 2 (A) Statistical chart of disease onset time after inoculation with Fusarium oxysporum, Alternaria ginseng, and Botrytis cinerea in Vietnamese ginseng treated with foliar fertilizer; (B) Diagram of lesion diameter 10 days after inoculation with Fusarium oxysporum in Vietnamese ginseng treated with foliar fertilizer; (C) Diagram of lesion diameter 10 days after inoculation with Alternaria ginseng in Vietnamese ginseng treated with foliar fertilizer; (D) Diagram of lesion diameter 10 days after inoculation with Botrytis cinerea in Vietnamese ginseng treated with foliar fertilizer.

[0016] Figure 3 Phenotypic images of Vietnamese ginseng treated with different foliar fertilizers 10 days after inoculation with Fusarium oxysporum, Alternaria ginseng, and Botrytis cinerea.

[0017] Figure 4 The graph shows the expression levels of disease resistance genes in Vietnamese ginseng under different foliar fertilizer treatments, where (A) represents the expression level of these genes. PvMPK6, PvPR1 and PvBIK Expression level graph; (B) PvPR3, PvBAK and PvTGA2 Expression level graph; (C) PvNPR, PvTGA1 and PvMPK3 Expression level graph; (D) PvFLS and PvPR2 Expression level graph; Note: For a single resistance gene, the bars from left to right represent the gene expression levels of Comparative Example 1, Comparative Example 5, Comparative Example 6, and Example 1. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] Example 1

[0020] A foliar fertilizer for enhancing the disease resistance of Vietnamese ginseng, by weight percentage, consists of 0.3% potassium dihydrogen phosphate, 0.1% boric acid, 0.5% urea, 0.01% chitosan, 0.0001% salicylic acid, and 0.05% Tween-20, with the balance being water.

[0021] Example 2

[0022] A foliar fertilizer for enhancing the disease resistance of Vietnamese ginseng, by weight percentage, consists of 0.1% potassium dihydrogen phosphate, 0.2% boric acid, 0.7% urea, 0.02% chitosan, 0.0001% salicylic acid, and 0.05% Tween-20, with the balance being water.

[0023] Example 3

[0024] A foliar fertilizer for enhancing the disease resistance of Vietnamese ginseng, by weight percentage, consists of 0.5% potassium dihydrogen phosphate, 0.1% boric acid, 0.3% urea, 0.005% chitosan, 0.0001% salicylic acid, and 0.05% Tween-20, with the remainder being water.

[0025] Comparative Example 1

[0026] Spray with an equal amount of water.

[0027] Comparative Example 2

[0028] The foliar fertilizer, by weight percentage, consists of 0.3% potassium dihydrogen phosphate, 0.1% boric acid, 0.5% urea, 0.0001% methyl jasmonate and 0.05% Tween-20, with the remainder being water.

[0029] Comparative Example 3

[0030] The foliar fertilizer, by weight percentage, consists of 0.3% potassium dihydrogen phosphate, 0.1% boric acid, 0.5% urea, 0.01% chitosan, and 0.05% Tween-20, with the remainder being water.

[0031] Comparative Example 4

[0032] The foliar fertilizer, by weight percentage, consists of 0.3% potassium dihydrogen phosphate, 0.1% boric acid, 0.5% urea, 0.0001% salicylic acid, and 0.05% Tween-20, with the remainder being water.

[0033] Comparative Example 5

[0034] The foliar fertilizer, by weight percentage, consists of 0.3% potassium dihydrogen phosphate, 0.1% boric acid, 0.5% urea, 0.01% chitosan, 0.00005% salicylic acid, and 0.05% Tween-20, with the remainder being water.

[0035] Comparative Example 6

[0036] The foliar fertilizer, by weight percentage, consists of 0.3% potassium dihydrogen phosphate, 0.1% boric acid, 0.5% urea, 0.01% chitosan, 0.0002% salicylic acid, and 0.05% Tween-20, with the remainder being water.

[0037] In the embodiments and comparative examples provided by this invention, the preparation method of the foliar fertilizer is as follows:

[0038] S1: Weigh out the chitosan powder and dissolve it in glacial acetic acid;

[0039] S2: Weigh out salicylic acid powder and dissolve it in anhydrous ethanol;

[0040] S3: Dissolve the appropriate amount of dissolved chitosan or salicylic acid, along with potassium dihydrogen phosphate, boric acid, urea, and Tween-20 in water according to the specific embodiment and comparative ratio, to prepare a foliar fertilizer of the required concentration.

[0041] Effect verification:

[0042] 1. Statistical analysis of lesion phenotypes

[0043] Three-year-old Vietnamese ginseng seedlings were selected and randomly divided into groups of three seedlings each. The foliar fertilizer solutions prepared in Example 1 and Comparative Examples 1-6 were sprayed onto the Vietnamese ginseng seedlings, with each treatment repeated three times. After seven consecutive days of spraying, Fusarium oxysporum cultured on PDA medium was collected using an inoculation needle. Fusarium oxysporum) Alternaria ginseng ( Alternariapanax ), Botrytis cinerea ( Botrytis cinerea ), and inoculated in the middle of the leaves of Vietnamese ginseng near the veins ( Figure 1 Cover the lesions with a bag to keep them moist. After obvious lesions appear, measure the diameter of the lesions, record the onset time, and observe the disease condition.

[0044] See ( Figure 2 A), the onset time in Example 1 was 7-9 days, significantly later than in Comparative Examples 1-6; see ( Figure 2The initial inoculation diameters of *Fusarium oxysporum*, *Alternaria ginseng*, and *Botrytis cinerea* in Example 1 were 0.6 cm. On day 10 after inoculation, the lesion diameters were 0.8 cm, 0.8 cm, and 0.7 cm, respectively, only slightly higher than the initial inoculation diameters of 0.2 cm, 0.2 cm, and 0.1 cm, and significantly lower than those in Comparative Examples 1-6. (See also...) Figure 3 The leaf lesion phenotype of Example 1 was significantly smaller than that of Comparative Examples 1-6, indicating that the disease resistance effect of Example 1 was better than that of the comparative examples.

[0045] 2. Enzyme activity test

[0046] Leaves from Examples 1, 1, 5, and 6, after inoculation, were harvested on day 10. The contents of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), ascorbic acid (ASA), reduced glutathione (GSH), and malondialdehyde (MDA) were measured using a kit. Comparative Example 1 served as the control group. The percentage increase or decrease in the defensive enzyme activity of Examples 1, 5, and 6 was calculated using the following formula.

[0047] Percentage increase in defense enzyme activity =

[0048] Percentage decrease in defense enzyme activity =

[0049] Table 1. Percentage increase or decrease in defensive enzymes after inoculation with Fusarium oxysporum.

[0050]

[0051] Table 2. Percentage increase or decrease in defensive enzymes after inoculation with *Alternaria ginseng*.

[0052]

[0053] Table 3 Percentage increase or decrease in defensive enzymes after inoculation with Staphylococcus aureus.

[0054]

[0055] Tables 1, 2, and 3 clearly show that regardless of whether the inoculation was with Fusarium oxysporum, Alternaria ginseng, or Botrytis cinerea, the percentage increase or decrease in enzyme activity in Example 1 was significantly higher than that in Comparative Examples 5 and 6, maximizing the disease resistance effect of Vietnamese ginseng.

[0056] 3. Resistance gene testing

[0057] Vietnamese ginseng was sprayed with foliar fertilizer solutions prepared according to Examples 1, 1, 5, and 6 for 7 consecutive days. Leaves were then harvested, and RNA was extracted using the RNAiso Plus kit (Takara, Japan). The RNA was then reverse transcribed into cDNA using the PrimeScript™ RT kit (containing 8×gDNA Eraser Premix, 5×RT Premix, RNase FreeH2O, Takara, Japan). Disease resistance genes for Vietnamese ginseng were designed. PvBIK, PvBAK, PvFLS PvMPK6, PvMPK3, PvPR3, PvPR2, PvPR1, PvTGA2, PvTGA1, PvNPR The primer sequences for the disease resistance gene and internal reference gene are shown in Table 4. Primer synthesis was commissioned to Sangon Biotech (Shanghai) Co., Ltd. Real-time quantitative PCR detection: CFX Connect was used. TM Real-time quantitative PCR was performed. The 20 µL reaction volume consisted of 1 µL template cDNA, 0.6 µL each of the primers, 10 µL TBGreen, and 7.8 µL PCR-grade pure water. The RT-PCR procedure was as follows: 95 °C pre-denaturation for 2 min; 95 °C denaturation for 5 s; 60 °C annealing for 30 s; 40 cycles. The relative gene expression level was calculated using the 2-ΔΔCT method.

[0058] The results showed that after 7 days of foliar fertilizer treatment, the foliar fertilizer content in the leaves of Vietnamese ginseng in Example 1 was significantly reduced. PvMPK6, PvPR1, PvBIK ( Figure 4 A), PvPR3, PvBAK, PvTGA2 ( Figure 4 B) PvNPR, PvTGA1, PvMPK3 ( Figure 4 C), PvFLS, PvPR2 ( Figure 4 D) The gene expression level was significantly higher than that of Comparative Examples 1, 5 and 6, indicating that the foliar fertilizer of Example 1 can maximize the expression of the defense enzyme gene, thereby enhancing the disease resistance of Vietnamese ginseng.

[0059] Table 4 Primer sequences of disease-resistant genes in Vietnamese ginseng

[0060]

[0061] In summary, the foliar fertilizer prepared in this invention has a significant effect on the prevention and control of diseases in Vietnamese ginseng. It has a positive effect on the prevention and control of Fusarium oxysporum, Alternaria ginseng, and Botrytis cinerea. Phenotypically, the use of the foliar fertilizer of this invention significantly inhibits the growth of pathogens, and the onset of disease in Vietnamese ginseng is significantly later than that in the control group. By testing the expression level of disease resistance genes in Vietnamese ginseng, the expression level of resistance genes in Vietnamese ginseng was significantly increased after the use of foliar fertilizer.

[0062] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a foliar fertilizer in enhancing the disease resistance of Vietnamese ginseng, characterized in that, The foliar fertilizer, by weight percentage, consists of 0.1–0.5% potassium dihydrogen phosphate, 0.1–0.2% boric acid, 0.3–0.7% urea, 0.005–0.02% chitosan, 0.0001% salicylic acid, and 0.05% Tween-20, with the balance being water; the application is to enhance the resistance of Vietnamese ginseng to diseases caused by Fusarium oxysporum, Alternaria ginseng, or Botrytis cinerea.

2. The application according to claim 1, characterized in that, The foliar fertilizer, by weight percentage, consists of 0.3% potassium dihydrogen phosphate, 0.1% boric acid, 0.5% urea, 0.01% chitosan, 0.0001% salicylic acid, and 0.05% Tween-20, with the balance being water.

3. The application according to claim 1, characterized in that, The application is to enhance the resistance of Vietnamese ginseng to root rot and black spot. Applications in diseases such as gray mold or gray mold.

4. The application according to claim 1 or 2, characterized in that, The method for preparing the foliar fertilizer is as follows: S1: Weigh out the chitosan powder and dissolve it in glacial acetic acid; S2: Weigh out salicylic acid powder and dissolve it in anhydrous ethanol; S3: Dissolve the appropriate amounts of dissolved chitosan and salicylic acid, as well as potassium dihydrogen phosphate, boric acid, urea, and Tween-20 in... Prepare foliar fertilizer at the required concentration in water.

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