Composition capable of inducing and resisting tobacco mosaic virus and application

Abstract

The invention is applied to the field of agriculture, and relates to a composition capable of inducing and resisting tobacco mosaic virus and application. The invention discloses a composition capable of inducing and resisting tobacco mosaic viruses. The composition is prepared from a tenebrio molitor extract, a seaweed extract and yeast nucleotide, wherein the mass ratio of the tenebrio molitor extract to the seaweed extract is (1-10): (1-10), and the concentration of yeast nucleotide in the composition is 50-2000mg / L. The tenebrio molitor extract, the seaweed extract and the yeast nucleotide are matched to play a synergistic role, the tobacco mosaic virus can be induced, resisted and prevented in a specific proportion, the quality and yield of agricultural products are improved, and the composition has an excellent synergistic effect.

Description

Technical Field

[0001] The invention is applied to the field of agriculture and relates to a composition capable of inducing resistance to tobacco mosaic virus and its application. Background Art

[0002] The immune mechanism in plants is like the vaccination of humans. After being treated with induction factors, the immune system in plants is quickly activated to defend against the infection of exogenous pathogens. That is, by using specific substances to stimulate the crop's own immune system and defense mechanism, crops can more effectively resist the infection process of pathogens. Plant immune induction technology can not only reduce the harm of crop diseases, but also improve the stress resistance of crops, such as cold resistance and drought resistance, while promoting crop growth and increasing yield and quality. Plant disease induction is a complex process involving multiple factors and mechanisms. Therefore, in practical applications, it is necessary to select appropriate induction technology and methods based on factors such as crop type, disease type and environmental conditions.

[0003] Tobacco mosaic virus (TMV) is an RNA virus belonging to the Tobamovirus group. It is the main pathogen of tobacco mosaic disease and is extremely harmful to tobacco. TMV virus particles are rod-shaped, about 300 nanometers by 15 nanometers in size, and consist of a single-stranded RNA and a protein shell. The RNA is wrapped in a shell formed by 2130 protein subunits. The virus is extremely stable and can survive in dry tobacco leaves for decades. It still has infectious activity after being diluted 1 million times. Tobacco mosaic virus can seriously damage the yield and quality of tobacco leaves, with a reduction of 20% to 80%. After the virus is infected, the growth of tobacco leaves is inhibited, and symptoms such as small leaves and deformities appear, resulting in the inability of tobacco to bloom and bear fruit normally, a small amount of seeds, uneven color of tobacco leaves after baking, poor smoke taste, and greatly reduced quality, which seriously affects the economic value of tobacco.

[0004] In the past, tobacco mosaic virus was mainly controlled by chemical pesticides, but the long-term use of chemical pesticides has led to increasingly obvious resistance and tolerance, and the problem of pesticide residues in the environment has become increasingly serious. Therefore, the development of biological agents to control plant diseases, such as tobacco mosaic virus, is a research hotspot in the agricultural field. Summary of the invention

[0005] The purpose of the present invention is to solve the problem that existing biological preparations are insufficiently applied in the agricultural field, and to provide a mixed preparation capable of inducing resistance to tobacco mosaic virus and its application.

[0006] To achieve the above object, the present invention adopts the technical solution as shown below.

[0007] A composition capable of inducing resistance to tobacco mosaic virus, comprising a yellow mealworm extract, a seaweed extract and yeast nucleotides; wherein the mass ratio of the yellow mealworm extract to the seaweed extract is 1-10:1-10, and the concentration of the yeast nucleotides in the composition is 50-2000 mg / L.

[0008] Preferably, the composition is a mealworm extract, a seaweed extract and yeast nucleotides; wherein the mass ratio of the mealworm extract to the seaweed extract is 1-5:1-3, and the concentration of yeast nucleotides in the composition is 100-600 mg / L.

[0009] The yellow mealworm extract is a brown viscous extract obtained by extracting yellow mealworms with ethanol, and is for standby use.

[0010] The mealworm extract is prepared by rapidly cooling the dried mealworms with liquid nitrogen and then grinding them, mixing them with ethanol and extracting them by reflux in a Soxhlet extractor for 6-8 hours, and concentrating the solution by vacuum distillation until the solution is a viscous brown paste, and volatilizing at room temperature to obtain the extract.

[0011] The dried mealworms are rapidly cooled and ground to obtain mealworm powder, which is then mixed with ethanol at a ratio of 1:20 (m / v).

[0012] The seaweed extract is obtained by extracting the algae of Sargassum fusiformis and / or Sargassum marinum; further, the product is derived from the algae of Sargassum fusiformis and Sargassum marinum, and contains polysaccharides, plant hormones, small molecule organic nutrients, vitamins and the like, and the substances used in the present invention are commercially available.

[0013] The yeast nucleotide is obtained by extracting high nucleotide tropical Candida as raw material through liquid deep fermentation. Further, the product is made by extracting high nucleotide tropical Candida as raw material through liquid deep fermentation, cell wall breaking, enzymolysis, separation and purification, and low-temperature drying, and is rich in nucleic acids, nucleotides, small peptides, polysaccharides and other substances, and the present invention uses commercially available ones.

[0014] An application of the composition capable of inducing resistance to tobacco mosaic virus, and the application of the composition in resisting tobacco mosaic virus.

[0015] A preparation for inducing resistance to tobacco mosaic virus contains the composition for inducing resistance to tobacco mosaic virus.

[0016] A preparation method of the preparation for inducing resistance to tobacco mosaic virus comprises the following steps: preparing a yellow mealworm extract and a seaweed extract into aqueous solutions respectively, and then mixing them in proportion, and then adding yeast nucleotides in proportion to obtain the preparation.

[0017] The advantages of the present invention are:

[0018] The present invention utilizes the combination of yellow mealworm extract, seaweed extract and yeast nucleotide to exert a synergistic effect, and under a specific ratio, can induce resistance to and prevent tobacco mosaic virus, improve the quality and yield of agricultural products, etc., and has an excellent synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram showing the relative expression levels of COI1, PR-1a and PR-1b in tobacco leaves after being treated with the composition of the present invention at different induction times. DETAILED DESCRIPTION

[0020] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0021] The tobacco variety used in the following examples is common tobacco NC89.

[0022] Example 1

[0023] The mealworm extract is prepared by adding liquid nitrogen to the dried mealworms, quickly grinding them into powder in a mortar, then mixing them with anhydrous ethanol at a ratio of 1:20 (m / v), and extracting them under reflux by heating in a Soxhlet extractor for 6 hours. The extract is concentrated to near dryness by reduced pressure distillation at 40°C using a rotary evaporator, and the concentrate is placed in a vacuum drying oven at 50°C and dried to constant weight to obtain a viscous brown extract. The ethanol (1:20) extraction rate of the extract is approximately 9.58%.

[0024] Application Examples

[0025] Inoculation of tobacco mosaic virus, disease index and determination of control effect

[0026] The tobacco variety is common tobacco NC89. The formula treatment is applied to tobacco plants with 5 to 6 leaves of the same growth, and the blank control is sprayed with water. The treatment group is sprayed with drugs according to the table below. After 24 hours of treatment, the samples of each group are inoculated with tobacco mosaic virus. The first leaf above the inoculated treatment leaf of each plant is inoculated by friction. The preparation method of inoculation venom is as follows: weigh 1g of fresh virus-carrying leaves, grind them with 100ml (pH 7.0) phosphate buffer, and then filter through multiple layers of gauze to obtain the filtrate. When inoculating, add a small amount of 60-mesh quartz sand, dip a small amount of inoculation liquid with your fingers, and gently rub the front of the tobacco leaves. The strength is just enough to cause a micro-wound on the leaf surface. Each treatment is repeated 3 times, and the inoculated plants are placed in a solar greenhouse for cultivation. Ten days after inoculation, the investigation is carried out according to the tobacco disease classification and investigation method (YC / T39-1996), and the disease index is calculated.

[0027] The relative levels of tobacco mosaic virus can be divided into levels 0, 1, 3, 5, 7, and 9, as follows.

[0028] Level 0: No visible symptoms;

[0029] Level 1: The heart leaves turn green or have bright veins;

[0030] Level 3: The heart leaves are obviously mosaic or wrinkled, and the old leaves are not diseased;

[0031] Level 5: The heart leaves are obviously mosaic or wrinkled, and other leaves are diseased and slightly uneven;

[0032] Level 7: The heart leaf is wrinkled and deformed, and other leaves are obviously mosaic;

[0033] Level 9: The leaves of the whole plant are mosaic, the leaves are wrinkled and deformed, and have dark green blisters.

[0034] According to the survey data, the disease index and control effect of each treatment are calculated. The disease index is calculated according to the following formula:

[0035]

[0036] Where:

[0037] X——disease index;

[0038] N i ——Number of diseased leaves at each level;

[0039] i——relative series;

[0040] N——Total number of leaves surveyed.

[0041] The control effect is calculated according to formula (2):

[0042]

[0043] Where:

[0044] P——control effect, in percentage (%);

[0045] CK——blank control disease index;

[0046] PT - Treatment index.

[0047] 1) Concentration optimization of different substances

[0048] The yellow mealworm extract, commercial seaweed extract, and commercial yeast nucleotide were prepared into aqueous solutions of 100 mg / L, 500 mg / L, 1000 mg / L, and 1500 mg / L to test the control effect of tobacco mosaic virus at different concentrations. The control was treated with clean water, and each treatment was repeated 3 times. The inoculation of tobacco mosaic virus, virus index, and control effect were determined as described above.

[0049] Table 1 Effects of different concentrations on the control of tobacco mosaic virus

[0050]

[0051] Note 1: P - control effect: 100-91% is A, 90-81% is B, 80-71% is C; 70-61% is D; 60-51% is E; 50-41% is F; 40-31% is G; 30-21% is H; 20-0% is I. Note 2: Seaweed extract was purchased from Henan Longteng Bioengineering Co., Ltd.; yeast nucleotides were purchased from Xi'an Tongze Biotechnology Co., Ltd.

[0052] From the above results, it can be seen that when the yellow mealworm extract, seaweed extract, and yeast nucleotide are applied alone to induce tobacco mosaic virus, their control effects are more obvious than the blank control. Among them, the control effect is best when the yellow mealworm extract is applied at a concentration of 1000 mg / L; the control effect is best when the seaweed extract is applied at a concentration of 1000 mg / L; and the control effect is best when the yeast nucleotide is applied at a concentration of 500 mg / L. Through the above concentration screening, the mixed formula of the biological agent will be screened with the optimal concentration of the above substances.

[0053] 2) Formula ratio optimization

[0054] According to the above record, the yellow mealworm extract solution and the seaweed extract solution were prepared respectively, and the yellow mealworm extract and the seaweed extract were mixed in a mass ratio of 3:1, 2:1, 1:1, 1:2, and 1:3, so that the final concentration of the mixed solution of the yellow mealworm extract and the seaweed extract was 1000 mg / L, and the concentration of yeast nucleotide in the mixed solution was ensured to be 500 mg / L, a blank treatment and three control treatments (control 1: a mixed solution of 1000 mg / L yellow mealworm extract and 500 mg / L yeast nucleotide; control 2: a mixed solution of 1000 mg / L seaweed extract and 500 mg / L yeast nucleotide; control 3: a mixed solution of yellow mealworm extract and seaweed extract with a final concentration of 1000 mg / L) were used to test the control effect of tobacco mosaic virus, and each treatment was repeated 3 times. The inoculation, virus index and control effect of tobacco mosaic virus were determined as described above, and the application amount per mu of land was 30 L / mu.

[0055] Table 2 Effect of different formula ratios on the control effect of tobacco mosaic virus

[0056]

[0057] As shown in Table 2, the control effects of all treatments were significantly higher than those of the blank control and each control. Among them, the best effect was achieved when the solution of 2:1 mealworm extract: seaweed extract (final concentration was 1000 mg / L) containing 500 mg / L of yeast nucleotide was applied, that is, its disease index was 6.6, and the control effect could reach 92.1%.

[0058] Example 3 Optimization of the time for inducing disease resistance

[0059] According to the treatment 2 described in Example 2, the composition was configured to conduct an experiment on the effect of induction time on the control effect of tobacco mosaic virus. A total of 4 different induction times (different times after spraying the combination) were set, 12, 24, 36, and 48 hours. The inoculation of tobacco mosaic virus, the virus index, and the control effect were determined as described above. After inoculation of tobacco mosaic virus, a gradient of induction time was set, and an induction time optimization test was conducted.

[0060] Table 3 Effect of induction time on the control effect of tobacco mosaic virus

[0061] Induction time Disease index Control effect (%) blank 83.2 0 12h 11.2 86.5 24h 6.6 92.1 36h 5.2 93.8 48h 9.1 89.1

[0062] As can be seen from Table 3, after applying the composition, as the induction time increases, the control effect of tobacco mosaic virus gradually increases. When the induction time is 36h, the control effect of tobacco mosaic virus is the best, reaching 93.8% of the control effect of tobacco mosaic virus, and then its control effect slowly decreases.

[0063] Example 4 Determination of relative gene expression

[0064] Tobacco leaves were collected at different induction times in the above examples, and the tobacco leaves were treated, while clean water was used as a blank, and each treatment was repeated three times.

[0065] Tobacco leaves were collected at 12, 24, 36, and 48 h of resistance, and each tobacco leaf was treated with liquid nitrogen and stored in a -80°C refrigerator. The relative expression levels of COI1 (Lu Zifeng et al., Grassland Science, 2014, 31(7): 1275-1282), a marker gene for systemic resistance mediated by plant disease resistance genes, and PR-1a and PR-1b (Li Lianhua, Guizhou University [D], 2015) in each leaf were detected (see Figure 1 ).

[0066] The detection method is

[0067] Total RNA was extracted according to the user manual of the TRIzol kit (Invitrogen, San Diego, CA) and purified according to the Super Smart cDNA Synthesis Kit (Takara, PrimeScript TM cDNA was reverse transcribed according to the instructions of SYBR Green I Master mix kit (Takara) using CFX Connect1M Real-Time System (Bio-Rad, USA). β-actin was used as an internal reference and 2 -△△CT The relative expression level was calculated by the gene-specific primers used in the present invention as follows:

[0068] for NtCOI1, 5′-TGCTTGACCGAGAGGAGAGA-3′ and 5′-CGCCCGACATAACTGAGACC-3′;

[0069] for acidic PR-1, 5′-GTCCATACTAATTGAAACGACCTAC-3′ and 5′-CCACTTCAGAGGATTACATATATAGTAC-3′;

[0070] For basic PR-1, 5'-TTTTGGTGGTATTATGGAGGTGTG-3' and 5'-ACAATTAACTGCCGTTGACTCATC-3';

[0071] β-actin (5'-GGTAACATTGTGCTCAGTGGTGG-3' and 5'-GGTGCAACGACCTTAATCTTCAT-3') was used as an internal control.

[0072] like Figure 1As shown, after being treated with the composition, with the increase of induction time (12, 24, 26, 48h), it was found that the expression of the key gene COI1 in the methyl jasmonate signaling pathway and the marker genes PR-1a and PR-1b in the systemic resistance mediated by plant disease resistance genes all showed an increasing trend. The relative expression of COI1 increased steadily, reaching 4.6 times at 36h. The expression of PR-1a and PR-1b genes increased compared with the control, and increased with the increase of induction time. At 36h, the relative expression of PR-1a and PR-1b genes was 5.9 times and 5.0 times that of the blank control. The data results of the above relative gene expression are consistent with the trend of the disease index and control effect of tobacco mosaic virus corresponding to the induction time. It is shown again that the optimal induction time of tobacco mosaic virus after applying the formula is 36h. This result also shows that the mixed formula of mealworm extract, seaweed extract and yeast nucleotide can regulate the relative gene expression levels of COI1, PR-1a and PR-1b to prevent and control TMV virus.

Claims

1. A composition having the ability to induce resistance to tobacco mosaic virus, characterized in that: The composition comprises a yellow mealworm extract, a seaweed extract and yeast nucleotides; wherein the mass ratio of the yellow mealworm extract to the seaweed extract is 1-10:1-10, and the concentration of the yeast nucleotides in the composition is 50-2000 mg / L.

2. The composition having the ability to resist tobacco mosaic virus according to claim 1, characterized in that: The composition comprises yellow mealworm extract, seaweed extract and yeast nucleotide; wherein the mass ratio of the yellow mealworm extract to the seaweed extract is 1-5:1-3, and the concentration of yeast nucleotide in the composition is 100-600 mg / L.

3. The composition having the ability to induce resistance to tobacco mosaic virus according to claim 1 or 2, characterized in that: The yellow mealworm extract is a brown viscous extract obtained by extracting yellow mealworms with ethanol, and is for standby use.

4. The composition having the ability to resist tobacco mosaic virus according to claim 3, characterized in that: The mealworm extract is prepared by rapidly cooling the dried mealworms with liquid nitrogen and then grinding them, mixing them with ethanol and extracting them by reflux in a Soxhlet extractor for 6-8 hours, and concentrating the solution by vacuum distillation until the solution is a viscous brown paste, and volatilizing at room temperature to obtain the extract.

5. The composition having the ability to resist tobacco mosaic virus according to claim 4, characterized in that: The dried mealworms are rapidly cooled and ground to obtain mealworm powder, which is then mixed with ethanol at a ratio of 1:20 (m / v).

6. The composition having the ability to induce resistance to tobacco mosaic virus according to claim 1 or 2, characterized in that: The seaweed extract is obtained by extracting the algae of Sargassum fusiformis and / or Sargassum marinum; The yeast nucleotide is obtained by extracting high nucleotide tropical Candida as a raw material through liquid deep fermentation.

7. Use of the composition having the ability to resist tobacco mosaic virus according to claim 1, characterized in that: The composition is used for resisting tobacco mosaic virus.

8. A preparation for inducing resistance to tobacco mosaic virus, characterized in that: A composition comprising the tobacco mosaic virus-resistant composition as claimed in claim 1.

9. A method for preparing the preparation for inducing resistance to tobacco mosaic virus according to claim 8, characterized in that: The yellow mealworm extract and the seaweed extract are prepared into an aqueous solution, and then mixed according to the proportion of claim 1, and yeast nucleotides are added according to the proportion after mixing to obtain the product.

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