Nano-composite for tomato virus disease prevention and control and whole-growth-period prevention and control method

Abstract

The invention provides a nano-composite for tomato virus disease prevention and control and a whole growth period prevention and control method. The nano-composite comprises an emodin monomethyl ether aqueous solution and a nano-carrier hydrophilic lipophilic diblock polymer HLDP in a mass ratio of 1: (10-20). According to the nanocomposite for tomato virus disease prevention and control, HLDP is added into an emodin methyl ether aqueous solution, and the HLDP and the emodin methyl ether aqueous solution are spontaneously combined through a hydrogen bond and Van der Waals' force, so that the particle size of an emodin methyl ether medicament is remarkably reduced to nanoscale size, and the affinity of the emodin methyl ether medicament to water is increased; the dispersibility in an aqueous solution and the wetting effect of the medicament on leaves are improved; meanwhile, the nano-composite has certain improvement on prevention and treatment effects on tomato spotted wilf virus, and the prevention effect is improved by about 11% compared with that of a single emodin monomethyl ether commodity medicament; compared with daily management measures of a base, the whole-growth-period field control effect of field seed dressing, root dipping and foliage spraying is adopted, and the morbidity of virus diseases is smaller than 10%; the tomato yield increase is promoted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agriculture, and particularly relates to a nanocomposite for prevention and control of tomato viral disease and a prevention and control method in the whole growth period. BACKGROUND

[0002] At present, there are more than 130 kinds of viruses that can infect tomatoes in the world, and about 40 kinds of viruses that can infect tomatoes have been reported in China, including cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), tomato spotted wilt virus (TSWV), tomato zonate spot virus (TZSV), tomato mosaic virus (ToMV), tomato chlorosis virus (ToCV) and tomato yellow leaf curl virus (TYLCV).

[0003] Plant viral disease is known as crop cancer, which is easy to spread, difficult to control and causes huge losses. At present, plant viral disease can be comprehensively prevented and controlled by chemical control, physical control, agronomic measures and some emerging technologies such as attenuated vaccine and gene editing, but there are problems such as low prevention and control efficiency, high cost and non-commercialization.

[0004] Nanomaterials have great application potential in the field of agriculture due to their unique structure and physicochemical properties: they can help pesticides penetrate leaf stomata or insect body wall, improve delivery efficiency; protect pesticides from decomposition in light or high temperature environment; improve the adhesion, wettability, adsorption, hydrophilicity and solubility of pesticides, promote plant uptake, significantly improve pesticide utilization efficiency and reduce dosage. Commonly used nanometer pesticide carriers mainly include porous hollow silica, nanoclay, metal and organic polymer materials, which can effectively embed and load pesticide active ingredients to nanoscale size, so that they have nanometer properties. At present, medicaments with particle size of 500 nm or less than 1000 nm are collectively referred to as nanometer pesticides; most of the existing nanomaterials have problems such as high cost, harsh use conditions and no safety evaluation on non-target organisms such as natural enemies of insects. SUMMARY

[0005] Therefore, the present application aims to overcome the defects in the prior art and provides a nanocomposite for prevention and control of tomato viral disease and a prevention and control method in the whole growth period.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] A nanocomposite for prevention and control of tomato viral disease, which comprises emodin methyl ether aqueous solution and nanocarrier hydrophilic-lipophilic double-polymer (HLDP) with a mass ratio of 1:10-20.

[0008] Preferably, the nanocomposite comprises emodin methyl ether aqueous solution and HLDP with a mass ratio of 1:16-20.

[0009] More preferably, the nanocomposite comprises methylellagic acid ether aqueous solution and HLDP at a mass ratio of 1:18.

[0010] Further, the methylellagic acid ether aqueous solution has a mass concentration of 0.1%.

[0011] The application also provides a nanocomposite medicament for preventing and controlling tomato virus disease, wherein the nanocomposite medicament comprises 0.1-1% of the nanocomposite.

[0012] Preferably, the nanocomposite medicament comprises 0.2-4% of the nanocomposite.

[0013] More preferably, the nanocomposite medicament comprises 0.2-0.35% of the nanocomposite.

[0014] The application also provides a whole growth period prevention and control method, comprising the following steps:

[0015] (1) using methylellagic acid ether medicament for seed dressing treatment before sowing;

[0016] (2) using methylellagic acid ether medicament for root dipping treatment on seedlings before planting;

[0017] (3) diluting the nanocomposite medicament of claim 4 or 5, and using the obtained diluent to spray the leaves of the plants after planting.

[0018] Further, the methylellagic acid ether medicament in step (1) is diluted from methylellagic acid ether aqueous solution; the methylellagic acid ether aqueous solution has a mass concentration of 0.1%; and the dilution ratio of the dilution step is 100-300 times.

[0019] Further, the methylellagic acid ether medicament in step (2) is diluted from methylellagic acid ether aqueous solution; the methylellagic acid ether aqueous solution has a mass concentration of 0.1%; and the dilution ratio of the dilution step is 400-600 times.

[0020] Further, the root dipping step in step (2) lasts for 15-20 seconds.

[0021] Further, the dilution ratio of the dilution step in step (3) is 400-600 times; and the spraying step in step (3) is specifically performed as follows: spraying once a week until the plants stably bear fruits.

[0022] Compared with the prior art, the application has the following advantages:

[0023] The nanocomposite for preventing and controlling tomato virus disease provided by the application is obtained by adding HLDP to emodin methyl ether aqueous solution, and the two spontaneously combine through hydrogen bonds and van der Waals forces, which significantly reduces the particle size of emodin methyl ether to nanoscale size, increases the affinity of emodin methyl ether to water, improves the dispersibility of emodin methyl ether in aqueous solution and the wetting effect of the medicament on leaves; meanwhile, the nanocomposite has certain promotion in the prevention and treatment effects on tomato tospovirus. The prevention effect is about 11% higher than that of the single dose of emodin methyl ether commercial medicament. The whole growth period prevention and control method provided by the application is based on the nanocomposite of emodin methyl ether and HLDP, and the nanocomposite is used for whole growth period prevention and control of tomato virus disease through seed dressing, seedling root dipping, foliage spraying during the growth period and the like. The results show that the nanocomposite can control the incidence of tomato virus disease to be below 10% in the field, and the incidence is at a low level, and the treatment effect is significant. In addition, the nanocomposite of emodin methyl ether / HLDP has no negative effect on the plant height and stem diameter of tomatoes, and is significantly better than the single dose of emodin methyl ether commercial medicament and the daily treatment in the base in terms of the fruit weight, diameter and the like. Therefore, the nanocomposite can be used as a tomato virus disease prevention and control medicament in agricultural production. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The schematic diagram of emodin methyl ether and HLDP in aqueous solution described in Embodiment 1 of the application is shown in Figure a, Figure b shows emodin methyl ether, and Figure c shows the comparison of emodin methyl ether and HLDP and emodin methyl ether;

[0025] Figure 2 The emodin methyl ether concentration and absorbance standard curve described in Embodiment 1 of the application are shown in Figure a and Figure b;

[0026] Figure 3 The particle size and morphology electron microscope photos of emodin methyl ether commercial medicament and the particle size and morphology electron microscope photos of the nanocomposite obtained by adding HLDP are shown in Figure a, Figure b and Figure c, wherein Figure a shows 0.1% emodin methyl ether commercial medicament (300 times dilution) without adding HLDP, Figure b shows the nanocomposite obtained by incubating and combining HLDP and 0.1% emodin methyl ether with a mass ratio of 1:1 (300 times dilution), Figure c shows the nanocomposite obtained by incubating and combining HLDP and 0.1% emodin methyl ether commercial medicament with a mass ratio of 5:1 (300 times dilution), and Figure d shows the nanocomposite obtained by incubating and combining HLDP and 0.1% emodin methyl ether commercial medicament with a mass ratio of 10:1 (300 times dilution);

[0027] Figure 4 The isothermal titration calorimetry determination of the incubation and combination of emodin methyl ether and HLDP described in Embodiment 1 of the application is shown in Figure a and Figure b;

[0028] Figure 5Symptoms and electrophoresis results of tobacco inoculated with TSWV (tomato spotted wilt virus) according to the embodiment 2 of the present application: wherein a is the electrophoresis result, and the negative control has no obvious band, and the amplified band of the treated group after inoculation is about 500 bp, which is the target product, and b is the tobacco after inoculation with TSWV, and it can be seen from the figure that uneven mottling appears on the leaves, which is the early symptom;

[0029] Figure 6 The real-time fluorescent quantitative primer standard curve according to the embodiment 2 of the present application;

[0030] Figure 7 Preventive and therapeutic effects of emodin methyl ether / HLDP complex on TSWV (tomato spotted wilt virus) according to the embodiment 2 of the present application: wherein a is the preventive effect, and a is the therapeutic effect; A is the water spraying treatment group, B is the HLDP solution with a content of 20 ug / mL, C is the 0.1% emodin methyl ether commercial drug diluted 500 times, and D is the nanometer complex of the self-combination of the 0.1% emodin methyl ether commercial drug diluted 500 times and HLDP;

[0031] Figure 8 Effects of emodin methyl ether / HLDP nanometer complex on tobacco growth according to the embodiment 2 of the present application: wherein a is the growth change rate of tobacco leaves, and b is the growth change rate of plant height; A, B and C are treatment types: A is the 0.1% emodin methyl ether commercial drug diluted 500 times treatment group, B is the emodin methyl ether / HLDP nanometer complex diluted 500 times treatment group, and C is the water control;

[0032] Figure 9 Transcriptome results according to the embodiment 2 of the present application: wherein a is the volcano plot of differential expression, b is the statistical result figure of KEGG enrichment of differential genes, and c is the heat map of differential gene expression amount;

[0033] Figure 10 Effects of emodin methyl ether / HLDP nanometer complex on tomato stem thickness according to the embodiment 3 of the present application: wherein a is 37 days after treatment, b is 45 days after treatment, and c is 53 days after treatment;

[0034] Figure 11 Effects of emodin methyl ether / HLDP nanometer complex on tomato plant height according to the embodiment 3 of the present application;

[0035] Figure 12 Promotion effects of emodin methyl ether / HLDP nanometer complex on tomato fruits according to the embodiment 3 of the present application: wherein a is 37 days after treatment, b is 45 days after treatment, and c is 53 days after treatment;

[0036] Figure 13The field control effect of the physcion / HLDP nanocomposite for whole growth period tomato virus disease described in embodiment 3 of the application: wherein, a figure is 30d after treatment, b figure is 40d after treatment, c figure is 50d after treatment. DETAILED DESCRIPTION

[0037] Except for the definition, the technical terms used in the following examples have the same meaning as generally understood by the person skilled in the art to which the application belongs. The test reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods, unless otherwise specified, are conventional methods.

[0038] The main reagents and materials involved in the experiment are as follows:

[0039] The synthesis method of lipophilic hydrophilic double block polymer (HLDP) is as follows:

[0040] (1) First, n-butanol (100 mg, 1.35 mmol), ε-caprolactone (6.16 g, 54 mmol) and stannous octoate (310 mg) were reacted in an oil bath at 90°C for 9 hours, dichloromethane (30 mL) was added to the mixture, and precipitated in excess cold methanol, and the precipitate was collected by filtration to obtain poly (caprolactone) (PCL);

[0041] (2) 2-bromoisobutyryl bromide (3 g, 13.04 mmol), tetrahydrofuran (30 mL) and triethylamine (4 g, 40 mmol) were added dropwise to the poly (caprolactone) solution (3 g, 0.58 mmol), and after the reaction was completed, methanol was added to terminate, and the precipitate was collected by filtration to obtain white powder of bromine-terminated poly (caprolactone) (PCL-Br);

[0042] (3) Bromine-terminated poly (caprolactone) (100 mg, 0.04 mmol) and dimethylaminoethyl methacrylate (0.87 g, 6.07 mmol) were dissolved in tetrahydrofuran (2 mL), and cuprous bromide (15 mg, 0.1 mmol) and pentamethyldiethylenetriamine (37 mg, 0.21 mmol) were added, and atom transfer radical polymerization was carried out at 65°C. After the reaction was completed, the crude product was purified by dialysis in water, and after freeze-drying, HLDP white powder was finally obtained.

[0043] 0.1% physcion aqueous solution: Inner Mongolia Qingyuan Baisheng Biotechnology Co., Ltd.

[0044] Physcion standard: Shanghai Aladdin Biochemical Technology Co., Ltd., purity > 98%.

[0045] 3% amino oligosaccharide: Shandong Haier Sanli Biological Chemical Co., Ltd.

[0046] Oligosaccharide · chain protein (Ate Ling): Hebei Zhongbao Green Crop Technology Co., Ltd.

[0047] Acetone (analytical pure): National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0048] Tobacco seeds: Nicotiana benthamiana.

[0049] Tomato seeds (Lola) origin: Israeli Hazera High Quality Seed Company; Importers: Beijing Oliwo Seed Industry Technology Co., Ltd.

[0050] Dialysis bag (regenerated cellulose, 1000Da): Shanghai Yuan Ye Biological Technology Co., Ltd.

[0051] FastPure Universal Plant Total RNA Isolation Kit (Plant Total RNA Rapid Extraction Kit RC411): Nanjing Novozyme Biological Technology Co., Ltd. PrimeScript RT reagent Kit with gDNAEraser (Perfect Real Time) (Reverse Transcription Kit, RR047A): TaKaRa, Japan.

[0052] 2x Taq Master Mix (Polymerase Chain Reaction Kit: Nanjing Novozyme Biological Technology Co., Ltd.

[0053] Taq Pro Universal SYBR qPCR Master Mix (SYBR Green Chimeric Real-time Fluorescent Quantitative Polynucleotide Chain Reaction Premix Kit): Nanjing Novozyme Biological Technology Co., Ltd.

[0054] The present application will be described in detail below in conjunction with examples.

[0055] Example 1 Loading efficiency and characterization of HLDP on emodin methyl ether

[0056] 1. Dispersing situation of emodin methyl ether standard and its HLDP nanocomposite in aqueous solution

[0057] The nanocarrier HLDP is a diblock polymer, which includes amine groups and bromide ions, long carbon chains, ester groups and other parts. The amine group is a hydrophilic part, the bromide ion group and the long carbon chain are hydrophobic parts, and the ester group also has relatively high affinity with water. According to the structural characteristics of emodin methyl ether and HLDP, the hydrophilic ability of emodin methyl ether will be enhanced after incubation and combination of the two.

[0058] The emodin methyl ether standard was mixed with the nano-carrier HLDP at a mass ratio of 1:10, and a 0.1 mg / mL emodin methyl ether aqueous solution was prepared. The solution was shaken to mix thoroughly, and then was left to stand at room temperature for 30 min to allow the emodin methyl ether to incubate and combine with the HLDP. An emodin methyl ether standard aqueous solution of the same concentration was also prepared for observation. Before observation, the solutions were shaken again to mix thoroughly. Figure 1 a is an emodin methyl ether / HLDP nano-complex aqueous solution, which has a high degree of dispersion in water and is relatively uniform. Figure 1 b is an emodin methyl ether single-agent aqueous solution, which shows that part of the solution is aggregated into a cluster in water, and the other part floats on the surface of the solution. Figure 1 c is a comparison of the two aqueous solutions after standing for 3 min. The emodin methyl ether single-agent forms a precipitate rapidly in the aqueous solution, and the other part floats on the surface of the solution or adheres to the wall of the test tube. The emodin methyl ether / HLDP nano-complex solution is still uniformly dispersed in water. The nano-carrier HLDP significantly increases the hydrophilicity of the emodin methyl ether.

[0059] 2. Loading efficiency of HLDP for emodin methyl ether

[0060] A standard curve of the concentration of emodin methyl ether was drawn by setting different concentration gradients, and a standard curve equation was calculated. The standard curve is shown in Figure 2 . Figure 2 The curve equation y is the concentration of emodin methyl ether (mg / mL), and x is the absorbance value. As can be seen from the equation, Figure 2 , a standard curve of the relationship between emodin methyl ether and absorbance value is y=0.031x, R 2 =0.9916, and the curve fitting is good. The absorbance value of the solution after dialysis is 0.358, and the concentration of emodin methyl ether is 0.011 mg / mL, and the concentration of HLDP is 0.22 mg / mL, according to the formula , the drug loading ratio is emodin methyl ether:HLDP=1:18, which is equivalent to a molar ratio of about 1:1 (the relative molecular mass of emodin methyl ether is 284.86, and the relative molecular mass of HLDP is 5136.3), i.e. 1 mol of emodin methyl ether can spontaneously combine with 1 mol of HLDP.

[0061] 3. Particle size of emodin methyl ether / HLDP complex

[0062] As shown in Table 1, when diluted 300 times, compared with the 0.1% emodin methyl ether aqueous solution commercial drug, the particle size of the HLDP nano-complex decreases with the increase of the mass ratio of the two. When the mass ratio is 1:10, the particle size decreases from 765.00 nm to 470.00 nm, which is reduced by about 63%, and the effect is the most significant.

[0063] Table 1 Particle size of emodin commercial drug and HLDP complex at 300 times dilution

[0064]

[0065] According to the results of scanning electron microscope, with the gradual increase of the mass ratio of nano-carrier HLDP, the particle size of emodin methyl ether / HLDP nano-complex gradually decreased, and the morphology changed greatly; which was consistent with the results of particle size analyzer.

[0066] 4. Thermodynamic characteristics of emodin methyl ether / HLDP nano-complex

[0067] As shown in Table 2, Figure 4 The data simulation results using Independent model showed that Kd(M) = 1.87 x 10 -5 , ΔH (kJ / mol) = -32.74, ΔS (J / mol·K) = -19.3, TΔS (kJ / mol) = 0.48, ΔG (kJ / mol) = -32.26. Among them, the Kd (dissociation constant) value was very low, indicating that emodin methyl ether and HLDP could be tightly combined; ΔG value was less than 0, indicating that emodin methyl ether and HLDP could be spontaneously combined at room temperature; and ΔH, ΔS were negative, indicating that they were combined by van der Waals force and hydrogen bonding force.

[0068] Table 2 Thermodynamic parameters of emodin methyl ether / HLDP nano-complex

[0069] Variable Value Kd(M) 1.87 x 10 -5 ]] ΔH(kJ / mol) -32.74 ΔS(J / mol·K) -19.3 TΔS(kJ / mol) 0.48 ΔG(kJ / mol) -32.26

[0070] 5. Contact angle analysis of emodin methyl ether / HLDP nano-complex

[0071] Table 3 Contact angle analysis of emodin methyl ether / HLDP nano-complex (glass)

[0072]

[0073]

[0074] Note: All treatments in the table (except water) are emodin effective ingredient content of 0.1% system, diluted 300 times; the letters "a, b, c" in the table represent whether there is significant difference, L and R are left contact angle and right contact angle.

[0075] As shown in Table 3, the contact angle of each treatment solution on glass showed a certain change rule: the contact angle decreased with the increase of the mass ratio of nano-carrier HLDP; the contact angle of commercial drug nano-complex was smaller than that of standard nano-complex.

[0076] Table 4 Contact angle analysis of physcion / HLDP nanocomposites on N. benthamiana

[0077]

[0078] Note: All solutions in the table are 0.1% physcion active ingredient system diluted 300 times.

[0079] From Table 4, the contact angle of each treatment solution on tobacco leaves showed a certain change rule, which decreased with the increase of the mass ratio of nanocarrier HLDP; the contact angle of commercial pesticides was smaller than that of the standard; the contact angle on the leaves was smaller than that on the glass.

[0080] 6, Physcion / HLDP nanocomposite drug retention and potential determination

[0081] Table 5 Physcion / HLDP nanocomposite drug retention determination

[0082]

[0083] From Table 5, the adhesion amount of physcion on the surface of N. benthamiana leaves increased with the increase of the mass ratio of HLDP.

[0084] Table 6 Potential of physcion and its HLDP nanocomposite in each treatment

[0085]

[0086] According to Table 6, in each treatment system, compared with physcion, the absolute value of the potential of the HLDP nanocomposite increased, especially the standard changed from negative potential to positive potential, indicating that the nanocarrier HLDP could make the drug system more stable. The surface of the leaves generally has a negative charge, if the drug has a positive charge, the two can be more closely combined through electrostatic interaction, thereby reducing the drift of the drug.

[0087] Example 2 Physcion / HLDP nanocomposite for prevention and control of TSWV (tomato spotted wilt virus)

[0088] 1. TSWV inoculation and propagation effect evaluation

[0089] Tobacco was inoculated with TSWV, and the upper diseased leaves were collected, RNA was extracted, reverse transcribed, and PCR was performed; the PCR products were subjected to agarose gel electrophoresis.

[0090] According to the electrophoresis results, Figure 5)It can be observed that the band is the target band. The PCR result is sequenced and put into NCBI for alignment. The alignment result shows that the similarity between the PCR product and the virus strain with the sequence number OQ627070.1 in the NCBI library is 99.34%, so it can be confirmed that both the inoculation virus and the disease virus are tomato spotted wilt virus.

[0091] 2, Preventive and therapeutic effects of emodin methyl ether / HLDP nanocomposite on TSWV virus

[0092] 0.1% emodin methyl ether commercial drug was mixed with nanocarrier HLDP at a mass ratio of 1:10, incubated, and then diluted to 500 times for standby.

[0093] (1) Therapeutic effect: 5-6 leaf age healthy tobacco with uniform growth was rubbed for inoculation; 24 hours after inoculation, the tobacco was randomly divided into 4 groups, 6 plants in each group, and sprayed with water (control group), 20 ug / mL of HLDP solution, 0.1% emodin methyl ether commercial drug 500 times liquid, and 0.1% emodin methyl ether commercial drug / HLDP nanocomposite 500 times liquid, respectively. After 6 days of application, the new leaves of Nicotiana benthamiana were cut to extract RNA, and reverse transcription and qPCR reaction were performed to detect the number of virus particles in the tobacco tissue; tobacco Actin gene was selected as the internal reference gene, and NP gene (nucleocapsid protein gene) in TSWV (tomato spotted wilt virus) was selected as the detection gene, and the relative expression amount of NP was calculated by 2 -ΔΔCT method.

[0094] (2) Preventive effect: A batch of healthy and uniform Nicotiana benthamiana was selected and randomly divided into 4 groups, 6 plants in each group, and sprayed with water, 20 ug / mL of HLDP solution, 0.1% emodin methyl ether commercial drug 500 times liquid, and 0.1% emodin methyl ether commercial drug / HLDP nanocomposite 500 times liquid, respectively, and then inoculated with TSWV 72 hours later. The top new leaves were taken for RNA extraction and subsequent tests 5 days after inoculation, and the steps and data processing were consistent with those in (1).

[0095] The standard curve is shown in Figure 6 , and two groups of primers were finally selected from the designed 5 internal reference gene primers and 13 target gene primers for the test; the relative expression amount of NP gene (nucleocapsid protein gene) was detected by qPCR, and the results are shown in Figure 7 .

[0096] As can be seen from Figure 7 , the content of anti-tomato spotted wilt virus NP gene (nucleocapsid protein gene) in tobacco sprayed with HLDP has no significant difference with the water control, and the nanocarrier HLDP has no therapeutic and preventive effect on tomato spotted wilt virus.

[0097] The treatment results show that the inhibition rate of emodin methyl ether / HLDP nanocomposite treatment on tomato virus expression is 81.45%, which is higher than that of 0.1% emodin methyl ether commercial agent single agent of 77.0%, and the control effect is increased by about 5%, which has a significant synergistic effect. The prevention results show that the inhibition rate of 0.1% emodin methyl ether / HLDP nanocomposite treatment on tomato virus expression is 81.75%, which is higher than that of the single agent of 73.84%, and the control effect is increased by 10.71%, which has a significant synergistic effect.

[0098] 3. Effect of emodin methyl ether / HLDP nanocomposite on tobacco growth

[0099] The effect of 0.1% emodin methyl ether commercial agent / HLDP nanocomposite on tobacco growth was analyzed to evaluate its safety.

[0100] Select 5-6 leaf stage N. benthamiana with normal growth, and randomly divide into 3 groups, 4-6 plants per group, and measure the leaf width and plant height of the 3rd, 4th and 5th leaves. Spray with water, 0.1% emodin methyl ether commercial agent 500 times liquid, and 0.1% emodin methyl ether commercial agent / HLDP nanocomposite 500 times liquid, respectively. Spray for 5 days, then measure the leaf width and plant height again, record and analyze the data to compare the growth rate.

[0101] The results are shown in Figure 8 There is no significant difference between the growth rate of tobacco leaf width and plant height treated with 0.1% emodin methyl ether single agent and 0.1% emodin methyl ether / HLDP nanocomposite and the water control; it has no effect on the growth phenotype of tobacco and is safe.

[0102] 4. Analysis of synergistic mechanism of emodin methyl ether / HLDP complex

[0103] Water, 0.1% emodin methyl ether commercial agent 500 times liquid, and 0.1% emodin methyl ether / HLDP nanocomposite 500 times liquid were used to spray 30-day-old tobacco, respectively. After 5 days, total RNA was extracted. After extraction, the quality of the RNA was detected, and a cDNA library was established. Sequencing was performed using the Illumina HiSeq platform. After removing low-quality Reads (including Reads with a proportion of N greater than 10% and Reads with a base number of quality value Q≤10 accounting for more than 50% of the whole Read), the sequencing data was aligned with the reference species gene library to obtain genomic location information and sequence characteristic information specific to the sequencing sample. After alignment, the transcript gene expression was quantified, and FPKM was used as an indicator to measure the transcript or gene expression level. |Fold Change|≥2 and FDR<0.01 were used as the standard for screening differentially expressed genes. The screened genes were annotated and enriched using GO and KEGG databases for differential gene annotation and enrichment analysis.

[0104] Results are shown in Figure 9 As shown in Figure 6, compared with single agent, the expression of 310 genes in tobacco leaves treated with emodin methyl ether / HLDP nanocomposites changed, of which 271 genes were up-regulated and 39 genes were down-regulated. Many genes were related to plant disease resistance pathways.

[0105] PTI5-L expression was up-regulated. Pti5 is a member of the Pti transcription factor family. Pti overexpressing plants showed increased phenylalanine ammonia lyase, catalase, peroxidase enzyme activity and reduced malondialdehyde content, which are all related to plant antiviral activity.

[0106] NAC35 and NAC94 expression was up-regulated. NAC family can improve resistance to viruses by participating in the regulation of plant hormone regulation such as JA (jasmonic acid), ABA (abscisic acid), SA (salicylic acid) and other signal pathways. In addition, studies have shown that NAC overexpression can interact with viruses, reducing viral particle amplification and replication.

[0107] WAK2 and NPK1 expression was up-regulated. The proteins encoded by these two genes can regulate plant innate immunity. Studies have shown that in tobacco with up-regulated NPK1, TMV virus particles were significantly reduced. NPK1 may also play a role in multiple MAPK cascades.

[0108] OsWRKY36 expression was up-regulated. The transcription factor WRKY encoded by OsWRKY36 plays an important role in the signal transduction of SA (salicylic acid) and JA (jasmonic acid). The salicylic acid and jasmonic acid signaling pathways are important ways for plants to respond to biotic and abiotic stress. In addition, WRKY70 plays an important role as a positive regulator of plant defense. WRKY41 can regulate the rise of anthocyanins in oilseed rape, and anthocyanins, as a kind of flavonoid substance, have good antioxidant properties and can improve resistance.

[0109] LRR-GSO1 and LRR-FS2 expression was up-regulated. These genes can regulate LRR expression. LRR is an important domain in plant receptor-like kinase proteins (RLK) that recognize pathogens, and also plays an important role in subsequent defense activation. The plasma membrane-localized FLAGELLIN SENSING 2 (FLS2) receptor is an important component of plant immunity to potential pathogenic bacteria, and can recognize the conserved flg22 peptide of flagellin. FLS2 has the common structure of transmembrane receptor kinases, with a receptor-like extracellular domain composed of leucine-rich repeat (LRR) and active intracellular kinase domain. After ligand binding, FLS2 dimerizes with the regulatory LRR receptor kinase BRI1-associated kinase 1, triggering downstream signal cascade and enhancing the disease resistance of the plant body.

[0110] ERF61 expression is up-regulated. ERF (ethylene response factor) plays an important role in plant disease resistance mechanism, and can act as a positive regulator to participate in the defense response of plants to pathogens; studies have also shown that ERF plays a role in plant immune response by regulating various hormone signaling molecules such as SA (salicylic acid), JA (jasmonic acid) and ET (ethylene). Among the ethylene transcription factors, ZmERF061 has recently been shown to be able to improve the resistance of corn to pathogenic bacteria by regulating the antioxidant defense system of plants.

[0111] In summary, compared with single agents, 0.1% physcion commercial drug / HLDP nanocomposite has a significant improvement in the prevention and treatment of tomato spotted wilt virus; 0.1% physcion / HLDP nanocomposite treatment of tobacco leaves, including PTI5-L, NAC35, NAC94, WAK2, NPK1, OsWRKY36, LRR-GSO1, LRR-FS2, ERF61 and other multiple plant disease resistance related pathway gene expression up-regulation, improve the resistance of tobacco to viral disease.

[0112] Example 3 Field verification of physcion and HLDP complex for prevention and treatment of tomato viral disease

[0113] The present application carries out field verification in the organic tomato base of Yuanmou County, Chuxiong Yi Autonomous Prefecture, Yunnan Province, evaluates the safety of physcion / HLDP nanocomposite to tomato and the prevention and control in the whole growth period, and compares the effect with the prevention and control methods of the currently commonly used amino oligosaccharide, oligosaccharide·chain protein and other agents.

[0114] 1. Safety evaluation of physcion / HLDP nanocomposite to tomato

[0115] The experiment has 9 treatments. Respectively: ① emodin methyl ether 400 times liquid foliar spraying; ② emodin methyl ether / HLDP(1∶18) nanocomposite 400 times liquid foliar spraying; ③ emodin methyl ether 500 times liquid root dipping+emodin methyl ether 400 times liquid foliar spraying; ④ emodin methyl ether 500 times liquid root dipping+emodin methyl ether / HLDP(1∶18) nanocomposite 400 times liquid foliar spraying; ⑤ emodin methyl ether 100 times liquid seed dressing+emodin methyl ether 400 times liquid foliar spraying; ⑥ emodin methyl ether 100 times liquid seed dressing+emodin methyl ether / HLDP(1∶18) nanocomposite 400 times liquid foliar spraying; ⑦ emodin methyl ether 100 times liquid seed dressing+emodin methyl ether 500 times liquid root dipping+emodin methyl ether 400 times liquid foliar spraying; ⑧ emodin methyl ether 100 times liquid seed dressing+emodin methyl ether 500 times liquid root dipping+emodin methyl ether / HLDP(1∶18) nanocomposite 400 times liquid foliar spraying; ⑨ base routine treatment (oligosaccharides · chain protein + amino oligosaccharin) 500 times spraying. Treatment ①-⑧ is carried out after three days of transplanting, and then once every 7 days, a total of 5 times, and then the same as ⑨. Each treatment has 3 repeats, and each repeat area is 60m 2 ; 37 days, 45 days, 53 days after treatment, the random sampling method is used to investigate the stem diameter and plant height of tomato by using vernier caliper and tape measure, and 10 plants are investigated in each repeat. The statistical results are shown in Figure 10 , Figure 11 .

[0116] The stem diameter results are shown in Figure 10 , and there is no significant difference between the stem diameter of tomato in each treatment of emodin methyl ether / HLDP nanocomposite and the base routine treatment; 45 days after treatment, the stem diameter of emodin methyl ether / HLDP(1∶18) nanocomposite 400 times liquid foliar spraying treatment is significantly higher than that of the base routine treatment.

[0117] The plant height results are shown in Figure 11 , and there is no significant difference between the plant height of tomato in each treatment of emodin methyl ether / HLDP nanocomposite and the base routine treatment 37 days and 45 days after treatment; in the last investigation (53 days after treatment), the plant height of tomato in each treatment of nanocomposite is significantly better than that of the base routine treatment.

[0118] In summary, emodin methyl ether / HLDP nanocomposite is safe for tomato and has no negative impact on the growth of tomato, and it is safe.

[0119] 2, Evaluation of the influence of emodin methyl ether / HLDP nanocomposite on tomato yield

[0120] The experiment designed 3 treatments. Respectively: ① emodin methyl ether 400 times liquid foliar spraying; ② emodin methyl ether / HLDP (1:18) nanocomposite 400 times liquid foliar spraying; ③ base routine treatment (oligosaccharide · chain protein + amino oligosaccharin) 500 times foliar spraying. Among them, ① and ② are sprayed at 12d, 20d, 28d after planting, and then the same as ③, sprayed once every 7 days; 3 repeats for each treatment, 60m 2 . At the harvest stage, the fruit quality was investigated by random sampling method, 5 plants for each treatment, 3 mature fruits were randomly selected from each plant, and the fruit diameter (vernier caliper) and single fruit weight (electronic balance), soluble solids content (Japan Atuo PAL-1) were measured and counted, and the results were as follows Figure 12 .

[0121] From Figure 12 it can be seen that emodin methyl ether / HLDP nanocomposite treatment has no significant effect on tomato fruit sugar content; single fruit weight and fruit diameter are significantly higher than emodin methyl ether commercial pesticide and base routine pesticide treatment. Emodin methyl ether / HLDP nanocomposite treatment has a certain promoting effect on tomato yield.

[0122] 3, Emodin methyl ether / HLDP nanocomposite tomato virus disease whole growth period field control effect

[0123] The experiment designed 5 treatments. Respectively: ① emodin methyl ether / HLDP (1:18) nanocomposite 400 times liquid foliar spraying; ② emodin methyl ether 500 times liquid root dipping + emodin methyl ether / HLDP (1:18) nanocomposite 400 times liquid foliar spraying; ③ emodin methyl ether 100 times liquid seed dressing + emodin methyl ether / HLDP (1:18) nanocomposite 400 times liquid foliar spraying; ④ emodin methyl ether 100 times liquid seed dressing + emodin methyl ether 500 times liquid root dipping + emodin methyl ether / HLDP (1:18) nanocomposite 400 times liquid foliar spraying; ⑤ base routine treatment (oligosaccharide · chain protein + amino oligosaccharin) 500 times spraying. 3d after transplanting, spray once every 7d, a total of 5 times; 3 repeats for each treatment, 60m 2 ; 30d, 40d, 50d after treatment, investigate and count the incidence. The whole growth period field tomato virus disease prevention and control effect is as follows Figure 13 .

[0124] From Figure 13 it can be seen that among the incidence investigated and counted, the incidence of emodin methyl ether / HLDP nanocomposite treatments (treatment ①-④) is significantly lower than that of base routine control (treatment ⑤), and in the later growth period of tomato, the incidence of the test group is not higher than 10%, which is much lower than that of base routine control (30%). Combined with the safety evaluation results, emodin methyl ether and HLDP composite can be used as one of the prevention and control agents for tomato virus disease in the field.

[0125] The above descriptions are only the preferred embodiment of the application, not intended to limit the application and any modification, equivalent replacement and improvement made within the principle and technical scope of the application should be included in the protection scope of the application.

Claims

1. A nanocomposite for the prevention and control of tomato viral diseases, characterized by: The nanocomposite comprises a physcion methyl ether aqueous solution and HLDP in a mass ratio of 1:10-20.

2. The nanocomposite for preventing and controlling tomato virus diseases according to claim 1, characterized in that: The nanocomposite comprises a physcion methyl ether aqueous solution and HLDP in a mass ratio of 1:16-20.

3. The nanocomposite for preventing and controlling tomato virus diseases according to claim 2, characterized in that: The mass concentration of the physcion methyl ether aqueous solution is 0.1%.

4. A nanocomposite agent for the prevention and control of tomato virus diseases, characterized by: The nanocomposite agent comprises 0.1-1% by mass of the nanocomposite according to any one of claims 1-3.

5. The method for preparing the nanocomposite agent for preventing and controlling tomato virus diseases according to claim 4, characterized in that: The nanocomposite medicament contains 0.2-4% of the nanocomposite by mass.

6. A method for prevention and control during the entire growth period, characterized by: The steps include: (1) Use physcion methyl ether to treat seeds before sowing; (2) Dip the roots of the seedlings with physcion before planting; (3) Diluting the nanocomposite agent according to claim 4 or 5, and spraying the resulting diluted solution on the leaves of the planted plants.

7. The whole growth period prevention and control method according to claim 6, characterized in that: The physcion methyl ether agent in the step (1) is prepared by diluting a physcion methyl ether aqueous solution; the mass concentration of the physcion methyl ether aqueous solution is 0.1%; and the dilution multiple in the dilution step is 100-300 times.

8. The whole growth period prevention and control method according to claim 6, characterized in that: The physcion methyl ether agent in the step (2) is prepared by diluting the physcion methyl ether aqueous solution; the mass concentration of the physcion methyl ether aqueous solution is 0.1%; and the dilution multiple in the dilution step is 400-600 times.

9. The whole growth period prevention and control method according to claim 6, characterized in that: The root dipping step in step (2) takes 15-20 seconds.

10. The whole growth period prevention and control method according to claim 6, characterized in that: The dilution ratio in the step (3) is 400-600 times; the specific operation of the spraying step in the step (3) is: spraying once a week until the plants stably set fruit.

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