Immune resistance inducer for preventing EAPV and TeMV and application thereof

Abstract

The invention belongs to the technical field of plant virus prevention and control, and particularly discloses an immune resistance inducer for preventing EAPV and TeMV and application of the immune resistance inducer, the immune resistance inducer is composed of a mixed aqueous solution of astragalus polysaccharide and an aluminum reagent, the application of the immune resistance inducer for preventing EAPV and TeMV in field prevention and control of EAPV and TeMV of passionflower is specifically that the immune resistance inducer is sprayed once in the seedling stage of 5-6 leaves of passionflower seedlings; and spraying once again at the initial stage of the growth period of the passion flower seedlings, namely when 12-14 leaves do not bloom. Compared with the prior art, the immune resistance inducer for preventing the EAPV and the TeMV has the advantages that the resistance inducing effect on the two main passionflower viruses and the growth promoting effect on the passionflower are better than those of the conventional commercial plant immune resistance inducer, namely altadine; in production, the Passiflora coerulea can be applied to prevention and control of virus diseases of the Passiflora coerulea in the seedling stage; the antiviral effect is efficient, and no harm is caused to plants; the method is low in cost and convenient for fruit farmers to operate, and has a strong application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of plant virus prevention and control, in particular to an immune inducer for preventing EAPV and TeMV and application thereof. Background Art

[0002] Passion fruit, scientifically known as Passiflora edulis, is a tropical and subtropical herbaceous vine plant, classified as passiflora in the family Passifloraceae. Passion fruit varieties in my country can be roughly divided into three types: yellow fruit, purple fruit and hybrid passion fruit. Passion fruit has extremely high economic value, and as an emerging fruit, its market demand is also increasing.

[0003] The growth cycle of passion fruit can be roughly divided into four stages: sowing, seedling, growth and maturity. After sowing, passion fruit seeds usually take about 1 month to germinate and emerge from the soil. After entering the seedling stage, the passion fruit seedlings will gradually grow true leaves and begin photosynthesis. As the growth period arrives, the passion fruit plants will gradually grow taller and begin to bloom and bear fruit. Finally, at the maturity stage, the passion fruit will become plump and bright in color, and can be picked and eaten at this time.

[0004] Passion fruit virus disease is one of the most serious diseases that harm passion fruit. The main viruses that infect passion fruit in China are: EAPV, TeMV, etc. (Huang Aijun et al., 2019; Yan Jiawen et al., 2018). Both EAPV and TeMV belong to the genus Potyvirus, positive single-stranded RNA viruses, and the virus particles are linear. The total length of EAPV is about 10.1kb, and the total length of TeMV is about 9.6kb. These two viruses infect passion fruit mainly causing appearance symptoms such as leaf mosaic, leaf shrinkage, deformity, and fruit lignification, and inhibiting the growth and development of the plant.

[0005] At present, the prevention and control measures for plant viral diseases mainly include planting disease-resistant varieties (Li Weihao, 2021), cultivating non-toxic seedlings, agricultural prevention and control measures (Lin Beisen, 2009), chemical prevention and control (Ma Xiaochun, 2022), plant immune inducer prevention and control methods (Jia Xiuling et al., 2016), etc.

[0006] Plant immune inducers are an environmentally friendly type of green pesticide that can enhance plant resistance and promote plant growth. They can be used as plant vaccines to induce the plant's own immune system for the purpose of plant disease resistance (Jia Xiuling et al., 2016). Common plant immune inducers include salicylic acid (SA), jasmonic acid (JA), benzothiadiazole (BTZ), etc. They all have the characteristics of long-lasting and stable resistance, broad spectrum, and pollution-free. According to different sources, plant immune inducers can be divided into biogenic elicitors and abiogenic active molecules. According to types, they can be divided into organic acids, inorganic compounds, oligosaccharides, protein polypeptides, and plant immune inducing bacteria: Trichoderma and Bacillus (Qiu Dewen, 2015).

[0007] Plant immune inducers do not directly inactivate pathogens. Their mechanism of action is to induce plants to activate their own immune systems, enabling plants to produce defense responses and promoting plant resistance to pathogens (Qiu D et al., 2017). Many plant immune inducers have a growth-promoting effect on plants, such as promoting root growth, cell division, bud differentiation, increasing tiller numbers and spikelet numbers. Physiological indicators such as plant height, stem girth, and root length of the treated plants are higher than those of plants under normal growth. Immune inducers usually increase the accumulation of plant growth and development-related hormones, improve the enzyme activity of plants, and enhance photosynthesis during the seedling stage of plants, thus enabling plants to grow better and faster (Wan Xuanwu et al., 2022).

[0008] There are already commercial immune inducer products on the market, and universities and companies at home and abroad are also actively researching and developing new immune inducers. Qiu Dewen (2015) developed a broad-spectrum immune inducer, oligosaccharide · streptoprotein preparation (Atailing), using the major protein elicitors PeaT1 and Hrip1 of Alternaria tenuissima. Spraying 500-fold dilution of 3% Alternaria tenuissima activating protein can significantly increase the seed setting rate, 1000-grain weight, and effective panicle number per hill of rice, and reduce the incidence of panicle neck blast (Sun Zhendong et al., 2020).

[0009] Astragalus polysaccharide is a water-soluble heteropolysaccharide extracted, concentrated, and purified from the dried roots of the leguminous plants Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao or Astragalus membranaceus (Fisch.) Bunge. It is composed of hexuronic acid, glucose, fructose, rhamnose, arabinose, galacturonic acid, glucuronic acid, etc. It can be used as an immune promoter or regulator, and at the same time has functions such as anti-tumor, anti-aging, anti-radiation, anti-stress, antioxidant, and anti-animal virus. Its antiviral principle is as follows: stimulating the functions of macrophages and T cells, increasing the number of E-ring-forming cells, inducing cytokines, promoting the induction of interleukin, and enabling the animal body to produce endogenous interferon, thereby achieving the purpose of antiviral. Song Yan et al. (2021) found that Astragalus polysaccharide can alleviate the symptoms of ulcerative colitis in mice by activating the adiponectin signaling pathway.

[0010] Aluminon is a yellowish-brown or reddish-brown powder, which is irritating to the skin. The molecular formula is C 22 H 23 N3O9, soluble in water, slightly soluble in ethanol, and almost insoluble in ether, acetone, and chloroform. The melting point is 220 - 225 °C (Shi Jianjun, 2010). Generally, it is used as a complexometric titration indicator to determine the content of aluminum in water, food, and tissues (Zhan Zongyong, 2017).

[0011] However, there is currently no precedent for the combined application of Astragalus polysaccharide and aluminon in the prevention and control of EAPV and TeMV. Summary of the Invention

[0012] To solve the above technical problems, the present invention provides an immune inducer for preventing EAPV and TeMV and its application.

[0013] To achieve the above object, the present invention is implemented according to the following technical scheme:

[0014] The first technical scheme of the present invention is an immune inducer for preventing EAPV and TeMV, which is composed of a mixed aqueous solution of Astragalus polysaccharide and aluminon. The concentration of Astragalus polysaccharide is 5.0 - 10 g / L, the concentration of aluminon is 5.0 - 10 g / L, and the molecular formula of aluminon is C 22 H 23 N3O9; wherein, EAPV is Passiflora morifolia virus, and TeMV is Telosma mosaic virus.

[0015] Preferably, the concentration of Astragalus polysaccharide is 10 g / L, and the concentration of aluminon is 10 g / L.

[0016] The second technical solution of the present invention is the application of an immune elicitor for preventing EAPV and TeMV in the field control of EAPV and TeMV in passion fruit. Specifically, the immune elicitor for preventing EAPV and TeMV is sprayed once during the seedling stage of passion fruit seedlings with 5 - 6 leaves; and then sprayed once again during the initial growth stage of passion fruit seedlings, that is, when there are 12 - 14 leaves and the seedlings have not flowered.

[0017] Compared with the prior art, the present invention first applies astragalus polysaccharide and aluminum reagent to the prevention and control of EAPV and TeMV. Its elicitor effect against these two main passion fruit viruses and its growth - promoting effect on passion fruit are better than those of the existing commercial plant immune elicitor, Atailing. In production, it can be applied to the prevention and control of passion fruit virus diseases, and also to the prevention and control of virus diseases in passion fruit seedlings. Its antiviral effect has the characteristics of high efficiency and harmlessness to plants; it has a low cost, is convenient for fruit farmers to operate, and has a strong application prospect. Description of the Drawings

[0018] Figure 1 The disease incidence of passion fruit plants 14 days after inoculating EAPV and TeMV with different treatments: a, treatment with formulation 1; b, treatment with clear water; c, treatment with formulation 4.

[0019] Figure 2 The disease incidence of passion fruit plants 14 days after inoculating EAPV and TeMV with different treatments: left, treatment with formulation 4; middle, treatment with Atailing; right, treatment with clear water.

[0020] Figure 3 The growth status of passion fruit seedlings 30 days after treatment with different agents: left, treatment with formulation 4; middle, treatment with Atailing; right, treatment with clear water.

[0021] Figure 4 The appearance symptoms of passion fruit plants in the field after treatment with formulation 4 agent: the cluster on the left is treated with clear water; the cluster on the right is treated with formulation 4. Detailed Embodiments

[0022] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further details the present invention in combination with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0023] The passion fruit seedlings used in the following examples were seedlings of Yunnan Golden Fruit, and the seeds were provided by the Plant Virus Research Laboratory of South China Agricultural University. The passion fruit plants were obtained by conventional seedling raising of passion fruit seeds and were detected to be virus-free. The tested viruses EAPV (IB strain) and TeMV (Guangxi isolate) were both preserved and provided by the Plant Virus Laboratory of South China Agricultural University. Astragalus polysaccharide was purchased from Shaanxi Junhe Biotechnology Co., Ltd., and aluminon was purchased from Guangdong Daxiao Chemical Industry Co., Ltd.

[0024] Example 1. Pot experiment

[0025] At the seedling stage of passion fruit with 5 - 6 leaves, the young leaves of the seedlings were sprayed with the medicaments shown in Table 1. Each medicament was used to treat 20 seedlings. After 24 hours of induction, the viruses EAPV and TeMV were inoculated respectively (virus inoculation concentration: EAPV was 4.08×10 7 copies / μL; TeMV was 7.33×10 7 copies / μL). Fourteen days after inoculation, the systemic leaves above the inoculated leaves were taken to extract RNA, and the expression level of the virus was detected by fluorescence quantitative PCR; 30 days after inoculation, the disease index of passion fruit plants was counted and the relative control effect was calculated. The disease resistance effects of different medicament formulations were analyzed and compared to screen out the medicament with the best effect for subsequent experiments. The water treatment was used as the negative control, and the treatment with Atailing (5.0 g / L) was used as the positive control. There is no official standard for the grading of passion fruit virus diseases at present, and a self-formulated grading standard was adopted. The specific content is as follows:

[0026] Grade 0: The whole plant is disease-free;

[0027] Grade 1: The new leaves are slightly wrinkled, showing mosaic and mottling. There is no obvious yellowing of other leaves. The plant is not significantly dwarfed;

[0028] Grade 3: One-third of the leaves show mosaic, yellow spots and pustules; some leaves show yellowing. The fruits do not show lignification. The plant is dwarfed and has weak growth vigor;

[0029] Grade 5: One-half of the leaves show mosaic, yellow spots and pustules; some leaves show yellowing; the leaves are wrinkled and deformed, the pericarp is mottled, and the fruits show lignification. The plant is significantly dwarfed and has weak growth vigor;

[0030] Grade 7: More than two-thirds of the leaves show mosaic and yellowing; the leaves are severely wrinkled and deformed, the pericarp is mottled, and the fruits show severe lignification. The plant is significantly dwarfed and has weak growth vigor;

[0031] The calculation formulas for the disease index and relative control effect are as follows:

[0032] Disease index = ∑(number of diseased plants at each level × the value of that disease level) / (total number of plants surveyed × the highest disease level value) × 100;

[0033] Relative control efficacy (%) = (disease index of control - disease index of treatment) / disease index of control × 100;

[0034] Another group of plants treated with the agent was normally cultured without virus inoculation. After 30 days, the plant height, root length, and total fresh weight of passion fruit were measured, and their averages were calculated. The growth-promoting effect of the agent on passion fruit was analyzed. The water treatment was used as the negative control, and the treatment with Atailing (5.0 g / L) was used as the positive control. The measurement method was carried out according to the conventional method.

[0035] Table 1 Composition and preparation of agents for treating passion fruit seedlings

[0036] Formulation Specific components Formulation 1 Astragalus polysaccharide (5.0 g / L) + Aluminon reagent (5.0 g / L), mixed and dissolved in water Formulation 2 Astragalus polysaccharide (5.0 g / L) + Aluminon reagent (10.0 g / L), mixed and dissolved in water Formulation 3 Astragalus polysaccharide (10.0 g / L) + Aluminon reagent (5.0 g / L), mixed and dissolved in water Formulation 4 Astragalus polysaccharide (10.0 g / L) + Aluminon reagent (10.0 g / L), mixed and dissolved in water Formulation 5 Astragalus polysaccharide (15.0 g / L) + Aluminon reagent (5.0 g / L), mixed and dissolved in water Formulation 6 Astragalus polysaccharide (15.0 g / L) + Aluminon reagent (10.0 g / L), mixed and dissolved in water Atailing 5.0 g / L, CK+ Clear water CK-

[0037] After 14 days of treatment with the agent, the virus content in the leaves of passion fruit plants is shown in Table 2.

[0038] Table 2 Statistics of virus content in passion fruit seedlings after 14 days of treatment with the agent

[0039]

[0040] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0041] Figure 1 and Figure 2 showed the disease incidence of passion fruit plants after 14 days of inoculation with EAPV and TeMV under different treatments. The experimental results showed that after treating passion fruit seedlings with Formulation 2, Formulation 3, and Formulation 4, strong virus resistance could be induced. Among them, Formulation 4 had the best induction effect, which was better than that of Atailing. After treating the plants with Formulation 4, the leaf surface of the plants was smooth, without shrinkage or pathological yellowing. While the control treated with water showed symptoms such as leaf shrinkage, vein color change, and yellowing. The induction effects of Formulation 1, Formulation 5, and Formulation 6 on passion fruit seedlings were not good.

[0042] After 30 days of treatment with the agent, the disease incidence of passion fruit plants with virus disease is shown in Table 3.

[0043] Table 3 Statistics of the disease incidence of passion fruit plants after treatment with the agent

[0044] Treatment Disease index Relative control efficacy (%) Formulation 1 64.35 7.58 Formulation 2 48.45 30.42 Formulation 3 46.78 32.82 Formulation 4 15.56 77.65 Formulation 5 42.78 38.56 Formulation 6 65.65 5.72 Clear water (CK-) 69.63 Atailing (CK+) 43.67 37.28

[0045] As can be seen from Table 3, after treating passion fruit seedlings with Formulation 2, Formulation 3, Formulation 4, and Formulation 5, virus resistance could be induced. Among them, Formulation 4 had the best induction effect, and the relative control efficacy was better than that of Atailing.

[0046] After 30 days of treatment with the agent, the growth of passion fruit plants is shown in Table 4. The growth status of passion fruit plants in the treatment group and the control group is shown in Figure 3 .

[0047] Table 4 Statistics on the growth of passion fruit plants after treatment with pesticides

[0048] Treatment Plant height (cm) Root length (cm) Fresh weight (g) Formulation 1 28.20±1.30d 11.96±1.10d 10.70±0.80c Formulation 2 30.04±1.05c 12.34±0.86c 12.52±0.68b Formulation 3 35.04±1.40a 13.46±1.34b 12.42±1.50b Formulation 4 35.53±2.82a 15.90±1.89a 13.80±0.65a Formulation 5 28.89±0.24d 12.28±0.56c 12.42±1.35b Formulation 6 31.45±1.23b 13.45±0.46b 13.50±1.35a Clear water (CK-) 27.50±1.06e 11.60±0.86d 10.50±0.23c Atailing (CK+) 30.20±1.40c 13.62±1.18b 12.72±0.84b

[0049] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0050] From Table 4 and Figure 3 It can be seen that formula 1-formula 6 have a certain growth-promoting effect on passion fruit seedlings. Among them, formula 4 has the best growth-promoting effect, which is better than the growth-promoting effect of Atailing. Therefore, formula 4 is used to treat passion fruit in subsequent field trials.

[0051] Example 2: Field experiment

[0052] Field experiments were conducted in a field where passion fruit is planted all year round at the Zengcheng teaching base of South China Agricultural University. The experimental area is 1.2 mu. Use bamboo to build a climbing rack for passion fruit; each row of racks is 2-2.5 meters high and 3-4 meters wide. Select healthy passion fruit plants that have been bred before and plant them in the field according to conventional cultivation methods. The row spacing is 3 meters and the plant spacing is 2 meters. A certain amount of space should be maintained to avoid being too dense or too sparse. Treat the passion fruit plants with the agent of formula 4 at different growth stages. The composition of the agent is shown in Table 1. The treatment schedule is shown in Table 5. 20 passion fruit seedlings were treated with each agent. Leaf samples were taken at the maturity stage of passion fruit, and the expression levels of EAPV and TeMV were detected by fluorescent quantitative PCR; the disease index of passion fruit plant virus diseases was counted and the relative prevention effect was calculated. Analyze and compare the effects of anti-EAPV and TeMV under different treatment methods.

[0053] Table 5 Field treatment methods

[0054]

[0055] Table 6 shows the occurrence of mosaic disease and virus content in passion fruit plants at the harvest stage under different treatments.

[0056] Table 6 The occurrence of mosaic disease and virus content in passion fruit plants at harvest time under different treatments

[0057]

[0058] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0059] As can be seen from Table 6, under different treatment methods, compared with the clear water control, for the second treatment method, the detection rate of virus-infected passion fruit plants was the lowest, and the contents of EAPV and TeMV were the lowest. The overall effect was better than that of the treatment with Atailing. The first and third treatment methods also had antiviral effects, and the overall effect was comparable to that of the treatment with Atailing.

[0060] Furthermore, the disease incidence of passion fruit plants under the second treatment method was investigated in detail, and the disease index and relative control efficacy were calculated. The specific data are shown in Table 7.

[0061] Table 7 Statistics of disease index and relative control efficacy of passion fruit plants against mosaic disease in the field under the second treatment method

[0062] Agent Disease index Relative control efficacy (%) <![CDATA[Formulation 4 > 28.26 62.43 Atailing 38.67 48.59 Clear water 75.22

[0063] As can be seen from Table 7, under the treatment of Formula 4, the disease index of passion fruit plants was the lowest and the relative control efficacy was the highest, which was better than the relevant data of Atailing. The external symptoms of passion fruit plants are shown in Figure 4 . The leaves of passion fruit plants treated with the agent were flat and smooth, with almost no disease spots. The leaves of the control passion fruit plants treated with clear water were wrinkled and yellowish, with obvious blister spots.

[0064] From the above pot experiments and field experiments, it can be seen that spraying the elicitor at the seedling bed stage can improve the resistance of passion fruit seedlings to EAPV and TeMV and reduce the virus-carrying rate of passion fruit seedlings. The greenhouse pot experiments of this study proved that after treating passion fruit seedlings with Formulas 2, 3, and 4, they all had elicitor effects and growth-promoting effects on EAPV and TeMV; among them, the effect of Formula 4 was the best, which was better than the treatment effect of Atailing. The application method of the preparation is to spray the surface of the plant leaves at the 5-6 leaf stage of passion fruit seedlings, and one treatment is enough. Treatments with Formulas 1-6 all had good growth-promoting effects on passion fruit plants; among them, the effect of Formula 4 was the best.

[0065] Spraying the elicitor in the field is the latest and most effective idea for resisting plant virus infection. It is directly related to the quality and yield of passion fruit, and the income of fruit farmers. The field experiments proved that among the three treatment methods of the preparation in the field, the second method had the best antiviral effect, and the first and third methods also had antiviral effects. The overall effect was comparable to that of the treatment with Atailing. In each treatment method, the antiviral effect of Formula 4 was the best, the disease index of passion fruit plants was the lowest, and the relative control efficacy was the highest, which was better than the treatment effect of Atailing. Therefore, the recommended application method of the preparation is the second method: a total of two treatments. Treat once at the seedling stage with 5-6 leaves; treat once at the initial growth stage (12-14 leaves, not flowering).

[0066] In summary, the preparation composed of two components, namely astragalus polysaccharide and aluminon reagent, is a very good immune inducer and antagonist against TEAPV and TeMV. Its comprehensive effect is better than that of the commercially available Atailing, and it has good commercial potential. The specific formulation and usage method of the preparation have been determined by the present invention.

[0067] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. An immune elicitor for preventing EAPV and TeMV, characterized in that, It is composed of an aqueous mixed solution of astragalus polysaccharide and aluminon reagent. The concentration of the astragalus polysaccharide is 5.0 - 10 g / L, and the concentration of the aluminon reagent is 5.0 - 10 g / L. The molecular formula of the aluminon reagent is C 22 H 23 N3O9; wherein, EAPV is Passiflora coccinea virus, and TeMV is Telosma mosaic virus.

2. The immune inducer for preventing EAPV and TeMV according to claim 1, characterized in that, The concentration of the astragalus polysaccharide is 10 g / L, and the concentration of the aluminum reagent is 10 g / L.

3. Application of an immune inducer for preventing EAPV and TeMV as described in claim 1 or 2 in the field control of EAPV and TeMV in passion fruit.

4. The application according to claim 3, characterized in that Spray the immune inducer for preventing EAPV and TeMV once during the seedling stage when the passion fruit seedlings have 5 - 6 leaves; and spray the immune inducer for preventing EAPV and TeMV again at the initial stage of the growth period of the passion fruit seedlings, that is, when there are 12 - 14 leaves and the seedlings have not flowered.

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