A method for improving the resistance of plants to viral diseases by using ferrochlorophyllide

Abstract

Aiming at the problem of the lack of control agents for plant virus diseases in current production, the present invention provides a method for using chlorophyllide iron to improve the resistance of plants to virus diseases, belonging to the field of plant protection in agricultural science. After plants are inoculated with plant viruses (including RNA viruses and DNA viruses), the leaves of the plants are sprayed with chlorophyllide iron at a certain concentration, and the drug is used continuously for 2 times. It is found that compared with the control group (spraying clear water), the resistance level of the plants treated with chlorophyllide iron at 400-1000 mg / L (preparation concentration, each gram of the preparation contains 0.2 mg of chlorophyllide iron, and the effective ingredient concentration is 0.08-0.2 mg / L) is significantly improved, the disease symptoms are significantly alleviated or eliminated, and the virus accumulation amount in the plants is significantly reduced. Among them, the effect of the application concentration of 1000 mg / L is the best. For the plants treated by this method, the resistance level to virus diseases is significantly improved. This method is simple, economical, green and efficient, and can be directly applied to agricultural production and related research fields, which is of great significance for efficiently and environmentally protecting the healthy growth of crops.

Description

Technical Field

[0001] The present invention relates to a method for improving the resistance of plants to viral diseases by using ferrochlorophyllide, and belongs to the field of plant protection in agricultural science. Background Art

[0002] Plant viral diseases are known as "plant cancers". They are numerous in variety, wide in host range, great in harm, and difficult to control. They are the second largest plant diseases after fungal diseases. Currently, more than 1,300 plant viruses have been reported, which harm more than 1,000 plants, resulting in a serious decline in the yield and quality of crops. According to incomplete statistics, the annual economic loss of crops caused by plant viral diseases globally is as high as $60 billion, posing a great threat to the safe production of crops. The adjustment of planting structure and climate change have led to the increasingly common occurrence of plant viral diseases in China, showing the characteristics of suddenness, explosiveness, and recurrence, seriously affecting food production and the safety of the supply chain of important agricultural products. At present, the prevention and control of plant viral diseases in production mostly rely on agricultural measures and disease-resistant varieties. However, there are still prominent problems such as the lack of resistant varieties and the degradation of resistance in the prevention and control of many plant viral diseases. Antiviral agents are still the most direct and effective means for preventing and controlling plant viral diseases. Currently, there is an extreme shortage of plant viral disease control agents. Although there are some antiviral agent products on the market, such as moroxydine hydrochloride, oligosaccharins, difenoconazole phosphate, and octhilinone, the types of plant viruses are complex and the crops infected and damaged are diverse. The proportion of registered antiviral agent products among all pesticide registered products is less than 0.5%. There are no registered antiviral agent products for many newly emerging or explosive viral diseases, resulting in the situation that there is no available drug for the prevention and control of some viral diseases in production.

[0003] Plant immune elicitor products mainly activate the plant immune system, regulate defense-related signals and metabolic pathways in plants, induce plants to produce resistance to biotic or abiotic stresses, and improve the disease resistance and stress resistance levels of plants. Therefore, plant immune elicitor products can enhance the resistance of crops to a variety of viruses, have good broad-spectrum properties, and have great application prospects in the field of enhancing the resistance of crops to virus infection, etc. They are important candidate drugs for the innovation of highly efficient and broad-spectrum antiviral agents.

[0004] Chlorophyll iron, ISO common name: iron chlorine e6, is a green, loose, powdery solid. It is a new type of natural plant growth regulator extracted from silkworm excrement, which can regulate crop growth. This agent is green and environmentally friendly, and safe to use. Chlorophyll iron can inhibit chlorophyllase, delay chlorophyll degradation, enhance photosynthesis, promote root growth, and enhance plant stress resistance. At present, large-scale application tests of chlorophyll iron have been carried out on crops such as rice, wheat, and rapeseed, all showing good growth promotion, regulation, and yield increase effects. At the same time, chlorophyll iron also shows certain effects on enhancing the stress resistance of crops to biological and abiotic stresses. Preliminary studies have shown that chlorophyll iron can be associated with some disease-resistant hormones in plants, and can induce the up-regulation of pathogen-related genes in Nicotiana benthamiana by affecting signal pathways such as salicylic acid (SA), brassinolide (BR), and abscisic acid (ABA). These results indicate that chlorophyll iron has great application potential in improving plant resistance to virus diseases, which requires further research and verification by us. This is of great significance for improving the prevention and control level of plant virus diseases in China and promoting the green and low-carbon development of the agricultural industry. Summary of the Invention

[0005] In view of the above research background, the present invention takes chlorophyll iron and plant viruses as the research objects. After tobacco is inoculated with plant viruses, the leaves of the plants are sprayed with chlorophyll iron at a certain concentration, and the medicine is used continuously for 2 times. The disease incidence and virus content of the plants are statistically analyzed. It is found that compared with the control group (treated with water), after treatment with chlorophyll iron at 400-1000 mg / L (formulation concentration, each gram of formulation contains 0.2 mg of active ingredient chlorophyll iron, and the effective ingredient concentration is 0.08-0.2 mg / L), the plants grow well, the disease symptoms are significantly reduced or eliminated, and the virus accumulation in the plants is significantly decreased. Among them, the effect of the application concentration of 1000 mg / L is the best. The present invention provides a method for using chlorophyll iron to improve plant resistance to virus diseases. By treating plant leaves within the effective concentration range using this method, the resistance of the plants to virus diseases is significantly improved. This method is simple, economical, green, and efficient, and can be directly applied to agricultural production and related research fields to more efficiently and environmentally protect the healthy growth of crops. Brief Description of the Drawings

[0006] Figure 1 : Symptoms of plants treated with different concentrations of chlorophyll iron after inoculation with Tomato mottle mosaic virus (ToMMV) (A: virus inoculation by rubbing, B: virus inoculation by agroinfiltration).

[0007] Figure 2 : Virus accumulation in plants treated with different concentrations of chlorophyll iron after agroinfiltration inoculation with ToMMV.

[0008] Figure 3: Symptoms of plants treated with chlorophyll iron after infiltration inoculation with Tomato yellow leaf curl virus (TYLCV).

[0009] Figure 4 : Virus accumulation in plants treated with chlorophyll iron after infiltration inoculation with TYLCV. Detailed implementation manners

[0010] To better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0011] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0012] The plant virus isolates used in the embodiments of the present invention are provided by the inventor's laboratory, and the rest of the materials, reagents, etc. can all be obtained through commercial channels.

[0013] Example 1: Experiment on chlorophyll iron improving the resistance of plants to Tomato mottled mosaic virus (RNA virus)

[0014] The effect of chlorophyll iron on improving the resistance of plants to virus diseases was evaluated by using the tobacco infection system with Tomato mottled mosaic virus (ToMMV), a member of the genus Tobamovirus.

[0015] 1. Medicaments and plant materials

[0016] The chlorophyll iron preparation was purchased from Nanjing Baite Biotechnology Company. Each gram of the preparation contains 0.2 mg of pure chlorophyll iron as the active ingredient, and the auxiliary agent is β-cyclodextrin. The tobacco used in the experiment was Nicotiana benthamiana. The seeds were evenly scattered on the surface of moist soil (a mixture of vermiculite and nutrient soil in a ratio of 3:1), and placed in a plant cultivation room at 28 °C for seed germination. After 7 days, plastic cups were filled with soil, and the tobacco seedlings were transplanted to the center of the cup and the surrounding soil was flattened. One seedling was transplanted into each cup. The plastic cups were placed in a water tray, and the soil was fully wetted through the holes at the bottom of the cup. The tobacco seedlings were grown in a plant cultivation room at 28 °C with a photoperiod of 16 h of light and 8 h of darkness. Water was applied once every 7 days before the 4-leaf stage and once every 4 days after that. Nutrient solution was supplemented once every week.

[0017] 2. Virus and inoculation method

[0018] The ToMMV isolate used in the experiment was collected from tomato samples in Shenyang City, Liaoning Province by the inventor's laboratory in 2015, and the purification of the virus isolate was achieved through the preparation of an infectious clone, named ToMMV-LN (Genbank accession number: MN853592). Tobacco plants were inoculated with ToMMV by the friction inoculation method and the Agrobacterium infiltration method.

[0019] 1) Friction inoculation method

[0020] Select tobacco at the 6-leaf stage for friction inoculation. Cut the stored leaves infected with ToMMV into pieces and put them into a mortar. Add liquid nitrogen and grind them into powder. Add an appropriate amount of PBS buffer for homogenization. Centrifuge at 6000g for 5 min, and the supernatant is the crude virus extract. Select one leaf per plant as the inoculated leaf. Sprinkle a small amount of carborundum on the leaf surface, drop the crude virus extract, and gently rub with fingers. 0.5 mL of the crude virus extract is used for each tobacco plant to complete the inoculation. Spray a small amount of water on the surface of the friction-inoculated leaves 4 hours later.

[0021] 2) Agrobacterium infiltration inoculation method

[0022] About 1 month after sowing, select tobacco plants with 3 - 4 large leaves and no flowering for infiltration inoculation. Oscillate and culture the Agrobacterium containing the ToMMV infectious clone prepared in the laboratory until the OD value of the bacterial liquid is 0.8 - 1.2. Centrifuge at 6000g for 6 min to collect the bacteria. Add the infiltration solution to adjust the OD value to 0.6. After standing at room temperature for 2 h, select the back of the thicker and flatter leaves and use a needleless syringe for infiltration. 0.5 mL of the infiltration bacterial liquid is used for each tobacco plant.

[0023] 3. Chemical treatment

[0024] Two days after inoculating the virus on the tobacco, prepare different concentrations of chlorophyllide iron (preparation concentrations of 1000, 400, 200 mg / L) and spray them evenly on the tobacco leaves. Four days later, apply the drug for the second time. The drug is applied twice in total, and clear water is sprayed as a control. Limited by the solubility of the drug (it cannot be effectively dissolved when exceeding 1000 mg / L), the maximum value of the chlorophyllide iron gradient concentration used in the experiment is 1000 mg / L, and higher concentrations are not set. In both inoculation methods, 10 tobacco plants are treated with each concentration, and the experiment is repeated three times.

[0025] 4. Disease incidence of plants under different treatments

[0026] Observe the plant symptoms 3 days after the second application of the drug (i.e., 9 days after inoculation). The results of friction inoculation of tobacco show ( Figure 1 A), the ToMMV virus shows extremely strong pathogenicity. The control plants show severe symptoms such as stunted growth and chlorotic and withered leaves, and the whole plant tends to die. However, the plants treated with chlorophyllide iron (1000 and 400 mg / L) can grow normally with normal leaf color. In particular, the resistance level of the plants treated with 1000 mg / L is significantly improved, and no obvious lesions appear. In addition, the plants treated with 200 mg / L have the same severe lesions and abnormal growth as the control. The results of Agrobacterium infiltration show ( Figure 1B). After infiltration inoculation, the pathogenicity of the virus was relatively mild. Obvious chlorotic yellowing appeared on the leaves of the control group plants. Compared with the control group, after treatment with chlorophyllide iron (1000 and 400 mg / L), the plants grew normally, the resistance level was significantly improved, and there were fewer faded leaves. Similarly, there was no obvious change in the resistance level of the plants treated with 200 mg / L chlorophyllide iron, and the symptoms were similar to those of the control group.

[0027] 5. Detection of virus accumulation in plants treated differently

[0028] Three days after the second drug application (i.e., 9 days after inoculation), take the tobacco leaves of each treatment group inoculated by infiltration above. Take the leaves of 4 plants in each treatment group as 4 replicates (the control plants showed severe lethality after rubbing inoculation with the virus and were no longer suitable for virus accumulation detection, so no analysis was carried out). Extract the total RNA of the leaves by the Trizol method, reverse transcribe to synthesize the first-strand cDNA, and then perform fluorescence quantitative PCR to detect the accumulation of ToMMV in the plants. Use the ToMMV CP gene as the detection target and the Nicotiana benthamiana UBC gene as the internal reference. The primers used are as follows: qToM-cp-F: 5’-CATTGCTGGGAACTTTCGAT-3’, qToM-cp-R: 5’-CAGGCCAACCCAGACATACT-3’, qUBC-F: 5’-TTTCGGTCCTGATGATACTCCC-3’, qUBC-R: 5’-CACAGAGCAAAGACTGGA TTGA-3’. The reaction system is 20 μL: 2×SYBR Green PCR Master Mix 10 μL, cDNA 1 μL, upstream primer 1 μL, downstream primer 1 μL, ddH 2 O 7 μL. The reaction conditions are pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 5 sec, annealing at 60°C for 15 sec, 40 cycles; melting curve analysis, from 60°C to 95°C, increasing at 0.1°C / sec. After obtaining the Ct values of each sample, use the 2 -ΔΔCt method to calculate the relative level of the CP gene in the samples of each treatment group. The results show ( Figure 2 ), after treatment with chlorophyllide iron (1000 and 400 mg / L), the virus accumulation in the plants was significantly reduced. Compared with the control group, the relative contents of the virus in the plants of the 1000 and 400 mg / L treatment groups decreased by 82.31% and 49.85% respectively. The relative content of the virus in the plants of the 200 mg / L treatment group was similar to that of the control group, with no significant difference.

[0029] Based on the above results, it is concluded that the application of chlorophyllide iron can improve the resistance of plants to virus diseases. The use concentration is 400 - 1000 mg / L (formulation concentration), and the concentration of 1000 mg / L has the best effect.

[0030] Example 2: Hemin iron improves the resistance of plants to Tomato yellow leaf curl virus (DNA virus)

[0031] The effect of hemin iron on improving the resistance of plants to virus diseases was evaluated using the tobacco system infected by the geminivirus that seriously harms vegetable production - Tomato yellow leaf curl virus (TYLCV).

[0032] 1. Agents and plant materials

[0033] The agent hemin iron and the plant material tobacco used were the same as in Example 1.

[0034] 2. Virus and inoculation method

[0035] The TYLCV isolate used in the experiment was collected from tomato disease samples in Xinghua City, Jiangsu Province by the inventor's laboratory in 2007, and the purification of the virus isolate was achieved through the preparation of an infectious clone, named TYLCV-XH2 (Genbank accession number: GU111505). The tobacco plants were inoculated with TYLCV using the agroinfiltration method with the infectious clone, and the method steps were the same as in Example 1, with 1 mL of infiltration bacterial solution used for each tobacco plant.

[0036] 3. Disease incidence and virus accumulation in plants after the application of hemin iron

[0037] Three days after the tobacco plants were inoculated with the virus, hemin iron at a concentration of 1000 mg / L (formulation concentration) was evenly sprayed on the tobacco leaves. After 7 days, the second application was carried out, with a total of 2 applications. Spraying clear water was used as a control. There were 10 tobacco plants in each of the treatment group and the control group, and the experiment was repeated three times. The symptoms of the plants were observed 5 days after the second application (i.e., 15 days after inoculation). The results showed ( Figure 3 ) that the leaves of the plants in the control group showed obvious chlorosis, yellowing, curling and other typical symptoms of TYLCV damage, while the resistance level of the plants treated with hemin iron (1000 mg / L) was significantly improved, and the chlorosis and yellowing symptoms of the leaves were significantly reduced. At the same time, the leaves of the treatment group and the control group were taken, with 4 plants in each group as 4 replicates, and RNA was extracted for fluorescence quantitative PCR to detect the TYLCV accumulation in the plants. Using the TYLCV CP gene as the detection target, the primers used were: qV1-F: 5’-CCGCAACCGTGAAGAATGAT-3’, qV1-R: 5’-CTCATACTTGGCTGCCTCCT-3’, and the reaction system and method were referred to Example 1. The results showed ( Figure 4 ) that the virus accumulation in the plants was significantly reduced after the treatment with hemin iron. Compared with the control group, the relative content of the virus decreased by 34.76%. This result also proves that the application of hemin iron can improve the resistance of plants to virus diseases.

[0038] The above embodiments do not limit the present invention in any form.

Claims

1. A method for improving the resistance of plants to viral diseases by using ferrochlorophyllide, which is characterized in that: Before the onset of viral diseases or at the initial stage of the onset, the leaves of plants are sprayed with ferrochlorophyllide. The concentration of the medicament preparation is 400 - 1000 mg / L, and each gram of the preparation contains 0.2 mg of ferrochlorophyllide. The medicament is used continuously for 2 times, so as to achieve the purpose of improving the resistance of plants to viral diseases and protecting the healthy growth of plants; the virus is tomato mottle mosaic virus or tomato yellow leaf curl virus.

2. The method according to claim 1, which is characterized in that the two consecutive applications of the medicament need to be spaced 4 - 10 days apart.

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