Application of cordyceps militaris germ extract in preparation of pesticide for resisting tobacco mosaic virus

Abstract

The invention discloses an application of a cordyceps militaris germ extract in preparation of an anti-tobacco mosaic virus pesticide, and belongs to the technical field of pesticides. The cordyceps militaris embryo extract has the effect of inhibiting activity on TMV, provides a new way for efficient utilization of cordyceps militaris embryos, promotes high-quality development of the cordyceps militaris cultivation industry, and has important production value and practical significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pesticides, in particular to application of a cordyceps militaris germ extract in preparation of a tobacco mosaic virus resistant pesticide. BACKGROUND

[0002] Tobacco mosaic virus (TMV) is a widely spread plant virus that mainly harms solanaceous crops such as tobacco, tomato, pepper and eggplant, and also poses a threat to other economic crops. The leaves of plants infected with TMV exhibit yellow and green interlaced mosaic symptoms, and in severe cases, the leaves are shriveled and deformed, and even necrotic; the virus interferes with the normal metabolism of plants, resulting in dwarfing, affecting photosynthesis and reducing yield; after infection, the fruits of crops such as tomato and pepper may become smaller and deformed, and the commodity value is greatly reduced. The virus disease caused by TMV shows an increasing trend in the incidence rate and severity year by year in China, and plant virus diseases have caused serious threats to agricultural production worldwide, affecting the growth, development, yield and quality of plants. The main methods to control TMV include the cultivation of disease-resistant varieties, the development of natural products and the use of chemically synthesized pesticides. Traditional prevention and control relies on disease-resistant varieties or chemical pesticides, and long-term use of chemical pesticides for prevention and control may lead to pesticide resistance and environmental pollution problems, therefore, it is urgent to develop efficient and low-toxicity antiviral preparations.

[0003] Cordyceps militaris germ is the remaining substrate of cordyceps militaris fruiting body cultivation, and the main components are unutilized culture medium (such as rice, wheat, silkworm chrysalis powder, etc.) and mycelium residues. Cordyceps militaris germ extract, i.e., cordyceps militaris cultivation residue extract, with the expansion of the cordyceps militaris industry, efficient utilization of the germ has become a problem to be solved. In recent years, the reuse of edible fungus chaff has become a research hotspot, and relevant research reports are emerging in an endless stream. There is no research on the use of cordyceps militaris germ to explore its antiviral effect on plants.

[0004] The development of green antiviral pesticides has become a realistic problem to be solved in the current agricultural industry. Cordyceps militaris germ is large in quantity and low in utilization rate, and the germ contains rich natural metabolites, which has great potential to be developed into antiviral pesticides. The new green pesticides developed based thereon will help the efficient development of agriculture and promote the sustainable development of the edible fungus industry.

[0005] Therefore, how to develop a cordyceps militaris germ extract for use in preparation of a tobacco mosaic virus resistant pesticide is a technical problem to be solved by those skilled in the art. SUMMARY

[0006] Therefore, the present application provides a cordyceps militaris germ extract for use in preparation of a tobacco mosaic virus resistant pesticide.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] Use of a cordyceps militaris embryo extract in the preparation of a pesticide against tobacco mosaic virus.

[0009] Further, the cordyceps militaris embryo extract contains 0.053 mg / mL cordycepin and 0.800 mg / mL crude polysaccharide.

[0010] Further, the preparation method of the cordyceps militaris embryo extract comprises the following steps:

[0011] (1) After the cordyceps militaris strain is activated by a PDA solid culture medium, a mycelium block is picked and inoculated into a liquid culture medium in a constant temperature oscillator for dark culture, to obtain a liquid strain;

[0012] (2) A tissue culture bottle is used, oat medium is filled in the bottle, sterilized, and cooled, then 5 mL of the liquid strain obtained in step (1) is inoculated into each bottle for dark culture, when the mycelium penetrates more than two-thirds of the bottle, the mycelium is scratched, and the mycelium is cultured under light after the scratching treatment, and the fruiting body is harvested;

[0013] (3) The remaining culture residues are dried, crushed, and sieved to obtain cordyceps militaris embryo powder;

[0014] (4) The cordyceps militaris embryo obtained in step (3) is mixed with ultrapure water for ultrasonic treatment, the ultrapure water is used for constant volume, centrifugation is performed, and the supernatant is collected to obtain a cordyceps militaris embryo extract.

[0015] Further, the specific steps of step (1) are as follows: after the cordyceps militaris strain is activated by a PDA (potato dextrose agar culture medium) solid culture medium, a mycelium block with a size of 0.5 cm 2 is picked and inoculated into 150 mL of liquid culture medium, and the liquid culture medium is cultured in a constant temperature oscillator at 22°C and 140 r / min in the dark for 5-6 days to obtain a liquid strain.

[0016] Further, the specific steps of step (2) are as follows: a tissue culture bottle with a capacity of 500 mL is used, oat medium is filled in the bottle, the oat medium comprises 30 g of oat and 50 mL of pure water, the oat medium is sterilized at 121°C for 25 min, and after cooling, 5 mL of the liquid strain obtained in step (1) is inoculated into each bottle for dark culture at 18°C for 8-9 days, when the mycelium penetrates more than two-thirds of the bottle, the mycelium is scratched, and the mycelium is transferred to a culture condition of a temperature of 20°C and a humidity of 80%-90% for light culture for 50 days after the scratching treatment, and the fruiting body is harvested.

[0017] Further, the specific steps of step (3) are as follows: the remaining culture residues are dried at 60°C, crushed, and sieved through a 80-mesh sieve to obtain cordyceps militaris embryo powder.

[0018] Further, the specific step of step (4) is: mixing the pupa cordyceps germ obtained in step (3) and ultrapure water according to a mass-volume ratio of 1 g:80 mL to perform ultrasonic treatment, the power of ultrasonic treatment is 80W, the temperature of ultrasonic treatment is 25 DEG C, the time of ultrasonic treatment is 3h, the solution is made to 100 mL with ultrapure water, 8000 r / min centrifugal 15 min, and the supernatant is collected to obtain the pupa cordyceps germ extract.

[0019] Further, the dosage form of the tobacco mosaic virus resistant pesticide is a liquid preparation, the liquid preparation is prepared by dissolving the pupa cordyceps germ extract in water, and the concentration of the pupa cordyceps germ extract in the tobacco mosaic virus resistant pesticide is 0.01-0.05 g / mL.

[0020] Further, the concentration of the pupa cordyceps germ extract in the tobacco mosaic virus resistant pesticide is 0.05 g / mL.

[0021] Further, the application time of the tobacco mosaic virus resistant pesticide is the 5-6 leaf stage of tobacco, and the application amount is to form a transparent and uniform liquid film on the leaf surface without local accumulation and dripping after spraying.

[0022] The beneficial effects of the present application are that the pupa cordyceps germ extract has inhibitory activity on TMV, provides a new way for high-efficiency utilization of pupa cordyceps germ, promotes the high-quality development of the pupa cordyceps cultivation industry, and has important production value and practical significance. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is the safety concentration screening result of the pupa cordyceps germ extract, wherein A is the growth of the Bornier tobacco after spraying different concentrations of germ embryo extract for 5 days; B is part of the indexes with significant difference after spraying different concentrations of germ embryo extract for 5 days.

[0024] Figure 2 It is the prevention effect result of 0.05 g / mL pupa cordyceps germ extract on TMV, wherein A is an experimental flowchart; B is the fluorescence brightness under ultraviolet light after spraying 0.05 g / mL germ embryo extract; C is the accumulation amount of TMV after spraying 0.05 g / mL germ embryo extract detected by Western blot.

[0025] Figure 3 It is the prevention effect of different concentrations of polysaccharide on TMV, wherein A is the fluorescence brightness of TMV-GFP under ultraviolet light after spraying different concentrations of germ embryo polysaccharide solution; B is the accumulation amount of TMV-GFP after spraying different concentrations of mycelium polysaccharide solution detected by Western blot; C is the fluorescence brightness of TMV-GFP under ultraviolet light after spraying different concentrations of mycelium polysaccharide solution; and D is the accumulation amount of TMV-GFP after spraying different concentrations of germ embryo polysaccharide solution detected by Western blot.

[0026] Figure 4 Figure 9 shows the TMV system resistance induced by the extract of Cordyceps militaris embryo and polysaccharide. Figure 9A shows the fluorescence intensity of TMV-GFP in the upper leaves of Nicotiana benthamiana treated with the extract of Cordyceps militaris embryo and polysaccharide. Figure 9B and 9C show the accumulation of TMV-GFP detected by Western blot and qRT-PCR, respectively.

[0027] Figure 5 Figure 10 shows the preventive effect of different concentrations of cordycepin on TMV. Figure 10A shows the content of cordycepin in CME determined by HPLC. Figure 10B shows the fluorescence intensity of TMV-GFP in the leaves of Nicotiana benthamiana treated with different concentrations of cordycepin. Figure 10C shows the accumulation of TMV-GFP protein in the inoculated leaves detected by Western blot.

[0028] Figure 6 Figure 11 shows the expression of defense-related genes and the activity of defense enzymes after spraying the extract of Cordyceps militaris embryo. Figure 11A shows the expression of PR1. Figure 11B shows the expression of PR2. Figure 11C shows the expression of ICS1. Figure 11D shows the activity of phenylalanine ammonia lyase (PAL). Figure 11E shows the activity of superoxide dismutase (SOD).

[0029] Figure 7 Figure 12 shows the effect of different solutions on the growth of tomato seedlings and Nicotiana benthamiana. Figure 12A shows the effect of CME root irrigation on the phenotype and growth of tomato. Figure 12B shows the plant height, stem diameter, dry weight, fresh weight, chlorophyll content, and carotenoid content of tomato determined after 5 times of continuous CME root irrigation. Figure 12C shows the effect of CME root irrigation on the phenotype and growth of Nicotiana benthamiana. Figure 12D shows the plant height, plant width, leaf width, fresh weight, and dry weight of Nicotiana benthamiana determined after 5 times of continuous CME root irrigation.

[0030] Figure 8 Figure 13 shows the standard curve for determining the content of crude polysaccharide of Cordyceps militaris embryo. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0032] Embodiment 1

[0033] Application of the extract of Cordyceps militaris embryo in preparing a pesticide against tobacco mosaic virus:

[0034] In the extract of Cordyceps militaris embryo, the content of cordycepin is 0.053 mg / mL, and the content of crude polysaccharide of Cordyceps militaris embryo is 0.800 mg / mL.

[0035] The preparation method of the cordyceps militaris germ extract includes the following steps:

[0036] (1) The cordyceps militaris strain (NYC01, purchased from Shandong Meiao Biological Engineering Co., Ltd.) is activated through PDA solid culture medium, and cultured in a 25°C incubator for one week, and the mycelium is grown to 2 / 3 of the plate. A 0.5 cm 2 size of the fungus block is picked into 150 mL of liquid culture medium (liquid culture medium formula: 15 g of proteose peptone, 20 g of glucose, 5 g of yeast powder, 2 g of potassium dihydrogen phosphate, 1 g of magnesium sulfate, and 1 L of pure water), and cultured in a constant temperature shaker at 22°C and 140 r / min in the dark for 5 days to obtain a liquid strain.

[0037] (2) A 500 mL tissue culture bottle is filled with oat medium, the oat medium includes 30 g of oat and 50 mL of pure water, sterilized at 121°C for 25 min, and after cooling, 5 mL of the liquid strain obtained in step (1) is inoculated into each bottle, and cultured in the dark at 18°C for 9 days. When the mycelium penetrates the bottom (the mycelium grows to the bottom of the oat medium) more than two-thirds, the mycelium is treated, and after the mycelium treatment, it is transferred to a temperature of 20°C and a humidity of 80% for light culture for 50 days, and then the fruiting body is harvested.

[0038] (3) The remaining culture residues are dried at 60°C for 24 h, crushed, and passed through an 80 mesh sieve to obtain cordyceps militaris germ powder.

[0039] (4) 1 g (accurate to 0.001) of the cordyceps militaris germ obtained in step (3) is added to a 250 mL conical flask with 80 mL of ultrapure water, and placed in an ultrasonic instrument for ultrasonic treatment. The power of the ultrasonic treatment is 80 W, the temperature of the ultrasonic treatment is 25°C, and the time of the ultrasonic treatment is 3 h. After taking out, the volume is made up to 100 mL with ultrapure water, and centrifuged at 8000 r / min for 15 min. The supernatant is collected to obtain the cordyceps militaris germ extract. 3 mL of CME is passed through a 0.22 μm microporous filter membrane, and the content of cordycepin in the solution is determined by a high performance liquid chromatograph to be 0.053 mg / mL.

[0040] The dosage form of the tobacco mosaic virus resistant pesticide is a liquid preparation, which is prepared by concentrating the cordyceps militaris germ extract. The concentration of the cordyceps militaris germ extract in the tobacco mosaic virus resistant pesticide is 0.05 g / mL.

[0041] The application time of the tobacco mosaic virus resistant pesticide is the 5-6 leaf stage of tobacco, and the application amount is to form a transparent and uniform liquid film on the leaf surface without local accumulation and dripping after spraying.

[0042] Example 2

[0043] Compared with Example 1, except that the concentration of Cordyceps militaris embryo extract in the tobacco mosaic virus resistant pesticide is 0.02 g / mL, other steps and parameters are the same as Example 1.

[0044] Example 3

[0045] Compared with Example 1, except that the concentration of Cordyceps militaris embryo extract in the tobacco mosaic virus resistant pesticide is 0.03 g / mL, other steps and parameters are the same as Example 1.

[0046] Example 4

[0047] Compared with Example 1, except that the concentration of Cordyceps militaris embryo extract in the tobacco mosaic virus resistant pesticide is 0.04 g / mL, other steps and parameters are the same as Example 1.

[0048] Example 5

[0049] Compared with Example 1, except that the concentration of Cordyceps militaris embryo extract in the tobacco mosaic virus resistant pesticide is 0.01 g / mL, other steps and parameters are the same as Example 1.

[0050] 1. Safety concentration screening of Cordyceps militaris embryo extract (CME)

[0051] Set five concentration gradients of 0.01 g / mL, 0.02 g / mL, 0.05 g / mL, 0.1 g / mL and 0.2 g / mL of embryo extract, and spray them on the eight-leaf-stage Nicotiana benthamiana, continuously for 5 days, and observe the effects of different concentrations on the growth of Nicotiana benthamiana.

[0052] Figure 1 The safety concentration screening results of Cordyceps militaris embryo extract, wherein Figure A is the growth of Nicotiana benthamiana after spraying different concentrations of embryo extract for 5 days; Figure B is the part of growth indicators of Nicotiana benthamiana with significant difference after spraying different concentrations of embryo extract for 5 days.

[0053] Select 4-6 leaf stage, uniform and robust Nicotiana tabacum seedlings, and continuously spray different concentrations of CME for 5 days, with water as control. Take photos to record plant phenotypes 24 h after the last treatment, and measure related growth indicators. The results show that the plants treated with 0.01-0.05 g / mL CME grow normally, and there is no significant difference with the control ( Figure 1 Figure A). When the concentration of CME is increased to 0.1 or 0.2 g / mL, the plant growth is significantly inhibited, showing dwarfing and leaf yellowing ( Figure 1 Figure A). In addition, the plant width, leaf width and chlorophyll content of the high-concentration CME treatment group are significantly lower than those of the control ( Figure 1 Figure B). In summary, the safe concentration range of CME foliar application to Nicotiana benthamiana is 0.01-0.05 g / mL.

[0054] The results showed that when the concentration of the bacterial embryo extract was 0.01-0.05 g / mL, it had no significant effect on the growth of Nicotiana bungeana.

[0055] 2. Evaluation of the preventive effect of bacterial embryo extract on TMV

[0056] Based on the effective concentration of the crude extract of bacterial embryos against TMV, a preventive experiment was conducted to explore the effect of the bacterial embryo extract.

[0057] Figure 2 The results of screening for effective concentrations of Cordyceps militaris mycelial extract against TMV are shown in Figure A, which is the experimental flowchart; Figure B shows the fluorescence intensity under UV light after spraying 0.05 g / mL of mycelial extract; and Figure C shows the accumulation of TMV after spraying 0.05 g / mL of mycelial extract as detected by Western blot.

[0058] To detect the antiviral activity of CME against plants, an inoculation experiment was conducted using GFP-tagged tobacco mosaic virus (TMV-GFP) on Nicotiana benthamiana. CME at a concentration of 0.05 g / mL was used for resistance determination: after spraying CME for 5 consecutive days, TMV-GFP was inoculated, and the TMV virus content was compared at 3 and 7 days post-inoculation. Figure 2 (Figure A). For example... Figure 2 As shown in Figure B, at 3 dpi, the GFP fluorescence signal in the CME-treated leaves was significantly lower than that in the negative control; at 7 dpi, the upper leaves of the control group showed obvious GFP fluorescence, while only a few upper leaves in the CME-treated group showed weak GFP fluorescence. Further Western blot analysis of TMV-GFP protein accumulation revealed a sharp decrease in virus content in the CME-treated plants compared to the control group, a result consistent with fluorescence observations under ultraviolet light. Figure 2 (Figure C). The above results indicate that CME can significantly reduce TMV infection.

[0059] The results showed that 0.05 g / mL CME had a significant preventive effect against TMV.

[0060] 3. Screening of anti-TMV active ingredients from bacterial embryo extracts - crude polysaccharides from bacterial embryos

[0061] Based on the preventive effect of bacterial embryo extract on TMV, the bacterial embryo extract was initially purified, and crude polysaccharides were extracted from the bacterial embryo using water extraction and alcohol precipitation. Five concentration gradient polysaccharide solutions of 0.1 mg / mL, 0.2 mg / mL, 1 mg / mL, 2 mg / mL and 5 mg / mL were set up and sprayed on six-leaf stage Nicotiana spp. to explore the control effect of different concentrations of embryo polysaccharide solutions on TMV.

[0062] Take the concentration of 10 mg / mL CME 5 mL, add 15 mL 95% anhydrous ethanol, in 4℃ refrigerator for 12 h. Alcohol after 8000 r / min centrifugation 20 min. Remove the supernatant, the precipitate was dried after drying, namely the fruiting body crude polysaccharide, precise 10 mL hot deionized water dissolved, namely the sample to be tested.

[0063] Standard curve:

[0064] 1. Preparation of glucose standard solution: accurately weigh 10 mg of anhydrous glucose, dissolve in ultrapure water and constant volume to 100 mL, get 0.1 mg / mL standard stock solution. Take 6 centrifuge tubes, add 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL standard stock solution respectively, add ultrapure water to 1.0 mL, get the concentration of 0, 20, 40, 60, 80, 100 μg / mL gradient standard solution. Add 0.5 mL 5% phenol solution to each centrifuge tube, shake gently, then quickly add 2.5 mL concentrated sulfuric acid (along the wall, avoid local overheating), shake and put into 60℃ constant temperature water bath for 20 min, take out and cool to room temperature.

[0065] The above reaction solution was moved into 96 well enzyme labeled plate respectively, 200 μL was added to each well. With "0 μg / mL" tube as blank control, the absorbance (OD value) of each well was detected at 490 nm wavelength. With glucose concentration (μg / mL) as abscissa, average OD value as ordinate, linear regression equation was fitted by Excel, y = 0.004x - 0.0132 (R2 = 0.997), Figure 8 For determination of the standard curve of the content of the embryo crude polysaccharide

[0066] Sample detection:

[0067] Take 1.0 mL of the treated sample solution, repeat the steps in the "standard curve preparation" (add phenol, concentrated sulfuric acid, water bath). The OD value of the sample solution was detected by enzyme labeled instrument.

[0068] Table 1 detection results

[0069]

[0070] Figure 3 For the preventive effect of different concentrations of cordyceps militaris embryo polysaccharide on TMV, A figure is the fluorescence intensity under ultraviolet light after spraying different concentrations of embryo polysaccharide solution; B figure is the accumulation of TMV-GFP after spraying different concentrations of embryo polysaccharide solution. C figure is the fluorescence intensity under ultraviolet light after spraying different concentrations of mycelium polysaccharide solution; D figure is the accumulation of TMV-GFP after spraying different concentrations of mycelium polysaccharide solution.

[0071] Since the CME medium is rich in polysaccharides from oat and C. militaris mycelium, we isolated the crude polysaccharides from CME (CRP) to determine whether the polysaccharides in CME are involved in the anti-TMV activity. We diluted CRP into five concentrations and sprayed them on the leaves of N. benthamiana, with distilled water as the negative control. The results showed that the green fluorescence signal of the leaves treated with 0.1 and 0.2 mg / mL CRP was significantly weaker than that of the negative control at 3 dpi (Fig. 1A). However, there was no significant difference in the GFP signal between the leaves treated with CRP at a concentration of 1 mg / mL or higher and the negative control (Fig. 1A). Western blot further confirmed that the accumulation of TMV virus in the leaves treated with CRP was significantly reduced at 3 dpi (Fig. 1B), indicating that CRP has anti-TMV activity. Figure 3 Figure 3 Since both the oat medium and mycelium in CME contain polysaccharides, we collected the C. militaris mycelium and extracted the polysaccharides (CMP) to determine whether CMP plays a key role in the anti-TMV activity. Leaf spraying experiments showed that the GFP fluorescence signal of the inoculated leaves treated with 0.1, 0.2, and 1 mg / mL CMP was significantly weaker than that of the negative control (Fig. 2C), and the accumulation of TMV-GFP protein was correspondingly reduced, consistent with the results of UV observation (Fig. 2D). When the concentration of CMP was 0.2 or 1 mg / mL, its anti-viral effect was comparable to or even better than that of the positive control, nimbimycin (NNM).

[0072] In summary, continuous leaf spraying with appropriate concentrations of CRP or CMP can significantly inhibit TMV. Figure 3 Figure 3 Based on the above results, we further explored whether spraying only the lower leaves could induce resistance in the upper unsprayed leaves. We selected 0.05 g / mL CME, 0.2 mg / mL CRP, and 0.2 mg / mL CMP for systemic resistance induction tests. The results showed that the green fluorescence intensity of the upper leaves of the plants treated with CME, CRP, and CMP was significantly lower than that of the control at 3 dpi (Fig. 3A). qRT-PCR and Western blot quantitative analysis showed that all three treatments significantly reduced the accumulation of TMV-GFP transcripts and proteins, with CMP having the best induction of resistance (Fig. 3B, C). The results indicated that CME, CRP, and CMP can resist viral infection by enhancing systemic resistance.

[0073] The results showed that a 0.2 mg / mL concentration of embryo polysaccharide solution had a significant inhibitory effect on TMV.

[0074] The results showed that a 0.2 mg / mL concentration of embryo polysaccharide solution had a significant inhibitory effect on TMV. Figure 4 Figure 4 The results showed that a 0.2 mg / mL concentration of embryo polysaccharide solution had a significant inhibitory effect on TMV. Figure 4 The results showed that a 0.2 mg / mL concentration of embryo polysaccharide solution had a significant inhibitory effect on TMV.

[0075] The results showed that a 0.2 mg / mL concentration of embryo polysaccharide solution had a significant inhibitory effect on TMV. The results showed that a 0.2 mg / mL concentration of embryo polysaccharide solution had a significant inhibitory effect on TMV.

[0076] ​​5. Screening of active components of TMV-resistant embryo extract - cordycepin

[0077] Based on the preventive and therapeutic effects of embryo extract on TMV, cordycepin was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a purity of ≥98%. Five concentration gradients of cordycepin solution, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, were set up and sprayed on six-leaf-stage Nicotiana benthamiana to explore the preventive effect of different concentrations of cordycepin solution on TMV.

[0078] Figure 5 For the preventive effect of different concentrations of cordycepin on TMV, Figure A is the HPLC determination of the content of cordycepin in CME, Figure B is the TMV infection of Nicotiana benthamiana leaves after treatment with different concentrations of CMP, and Figure C is the Western blot detection of TMV-GFP protein accumulation in inoculated leaves.

[0079] To further verify the antiviral activity of cordycepin, the content of cordycepin in CME was determined by HPLC, and the results showed that the concentration of cordycepin in 0.05 g / mL CME solution was 0.053 mg / mL (Figure A of Figure 5 Accordingly, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL cordycepin standard solution was prepared and sprayed on Nicotiana benthamiana to evaluate its antiviral effect. After 5 days of continuous treatment, TMV-GFP was inoculated. The results showed that the fluorescence intensity of the inoculated leaves treated with 0.3 mg / mL cordycepin (Cor) was significantly lower than that of the negative control, and was comparable to that of the NNM treatment group; when the concentration was ≤0.2 mg / mL, there was no significant difference in GFP fluorescence intensity between the treatment group and the negative control. Notably, Cor at a concentration of ≥0.4 mg / mL caused obvious damage to the leaves and inhibited the growth of Nicotiana benthamiana (Figure B of Figure 5 Western blot further confirmed that 0.3 mg / mL Cor had the best preventive effect on TMV (Figure C of Figure 5 The results showed that cordycepin solution at a concentration of 0.3 mg / mL had a significant inhibitory effect on TMV.

[0080] 6. qRT-PCR verification of defense-related gene expression and defense enzyme activity determination

[0081] Figure 6 For the expression of defense-related genes after spraying embryo extract, Figure A is the expression of PR1, Figure B is the expression of PR2, and Figure C is the expression of ICS1. Figure D is the activity of phenylalanine ammonia lyase (PAL), and Figure E is the activity of superoxide dismutase (SOD).

[0082] It has been reported that many phytohormone-related pathways are involved in plant antiviral processes (such as salicylic acid, ethylene, jasmonic acid, etc.). To investigate whether the embryo extract enhances the resistance of plants to disease, we detected the key genes PR1 and PR2 of the disease-related protein and the gene ICS1 of the salicylic acid pathway by qRT-PCR. The resistance of plants is significantly related to the increase of the activity of defense-related enzymes. Therefore, we measured the activities of phenylalanine ammonia lyase (PAL) and superoxide dismutase (SOD) in tobacco leaves treated with different compounds (including CME, CRP, CMP, and Cor).

[0083] The results showed that the expression of disease-related proteins PR1, PR2, and ICS1 was significantly increased by spraying Cordyceps militaris embryo extract, and the activities of PAL and SOD were increased to different degrees before and after TMV inoculation. These results further confirmed that Cordyceps militaris embryo extract can enhance the resistance of plants to disease.

[0084] 7. Effects of root irrigation with Cordyceps militaris embryo extract on the growth of tomato seedlings and Nicotiana benthamiana

[0085] Tomato and Nicotiana benthamiana were treated with root irrigation with Cordyceps militaris embryo extract, water, and Huamodu nutrient solution as controls.

[0086] Figure 7 Effects of root irrigation with different solutions on the growth of Nicotiana benthamiana. Panel A shows the effects of CME root irrigation on the phenotype and growth of tomato, Panel B shows the plant height, stem diameter, dry weight, fresh weight, chlorophyll content, and carotenoid content measured after 5 consecutive CME root irrigations, Panel C shows the effects of CME root irrigation on the phenotype and growth of Nicotiana benthamiana, and Panel D shows the plant height, plant width, leaf width, fresh weight, and dry weight measured after 5 consecutive CME root irrigations.

[0087] To evaluate whether CME has a growth-promoting effect, we treated tomato seedlings with different concentrations of CME for 5 consecutive root irrigations, and then measured the plant height, stem diameter, dry weight, fresh weight, chlorophyll content, and carotenoid content. The results showed that there was no significant difference between the 1 mg / mL and 5 mg / mL CME treatment groups and the control group, while the above-mentioned indicators of the 10 mg / mL CME treatment group were significantly higher than those of the control group (Panels A and B of Fig. 6), indicating that this concentration has a significant growth-promoting effect on tomato. Figure 7 Figure 7

[0088] ​​In view of the oat component in CME raw materials, to verify whether the growth-promoting effect is mainly derived from the metabolites produced during the growth of Cordyceps militaris, we set up oat extract (OE) treatment group at the same time, and water as negative control, nutrient solution (NS) as positive control, to explore the growth-promoting effect of 10 mg / mL CME on Nicotiana benthamiana. The results showed that compared with the negative control, CME significantly increased the plant width, leaf width, dry weight and fresh weight of Nicotiana benthamiana; while the growth indicators of OE treatment group had no significant difference with the negative control Figure 7 the C graph of FIG. 1, Figure 7 the D graph of FIG. 1). The above results show that 10 mg / mL CME has a significant growth-promoting effect on plants, and the effect mainly comes from the metabolites produced during the growth of Cordyceps militaris.

[0089] The results show that the tomato treated with the germ extract solution has higher plant height, stem diameter, dry weight, fresh weight, chlorophyll and carotenoid than the control group. The growth of Nicotiana benthamiana treated with the germ extract solution is significantly better than that of the water and oat extract treatment groups, which can significantly improve the plant width, leaf width and fresh weight of Nicotiana benthamiana.

[0090] The above description of disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a Cordyceps militaris embryo extract in the preparation of a tobacco mosaic virus-resistant pesticide.

2. The use of a Cordyceps militaris embryo extract according to claim 1 for the preparation of a pesticide against tobacco mosaic virus, characterized in that, The content of cordycepin in the Cordyceps militaris embryo extract is 0.053 mg / mL, and the content of crude polysaccharide in the embryo is 0.800 mg / mL.

3. Use of the Cordyceps militaris embryo extract according to claim 1 for the preparation of a pesticide against tobacco mosaic virus, characterized in that, The preparation method of the Cordyceps militaris embryo extract comprises the following steps: (1) After the Cordyceps militaris strain is activated by PDA solid culture medium, the mycelium is inoculated into a liquid culture medium in a constant-temperature oscillator and cultured in the dark to obtain a liquid strain; (2) A tissue culture bottle is used, oat medium is filled in the bottle, sterilized, and cooled, and then 5 mL of the liquid strain obtained in step (1) is inoculated into each bottle for dark culture, and when the mycelium penetrates through more than two-thirds of the bottle, the mycelium is scratched, and the mycelium is cultured under light after scratching, and the fruiting bodies are harvested after 50 days of culture; (3) The remaining culture residues are dried, crushed, and sieved to obtain Cordyceps militaris embryo powder; (4) The Cordyceps militaris embryo obtained in step (3) is mixed with ultrapure water for ultrasonic treatment, the solution is diluted with ultrapure water, centrifuged, and the supernatant is collected to obtain the Cordyceps militaris embryo extract.

4. The use of a Cordyceps militaris embryo extract according to claim 3 for the preparation of a pesticide against tobacco mosaic virus, characterized in that, The specific steps of step (1) are: after the Cordyceps militaris strain is activated by PDA solid culture medium, a 0.5 cm 2 size of the fungus is picked into 150 mL of liquid culture medium, and cultured in a constant temperature oscillator at 22°C, 140 r / min for 5-6 days in the dark to obtain a liquid strain.

5. The use of a Cordyceps militaris embryo extract according to claim 3 for the preparation of a pesticide against tobacco mosaic virus, characterized in that, The specific steps of step (2) are as follows: a 500 mL tissue culture bottle is used, oat medium is filled in the bottle, the oat medium comprises 30 g of oat and 50 mL of pure water, and the medium is sterilized at 121°C for 25 min, and then 5 mL of the liquid strain obtained in step (1) is inoculated into each bottle for dark culture at 18°C for 8-9 days, and when the mycelium penetrates through more than two-thirds of the bottle, the mycelium is scratched, and the mycelium is cultured under light at a temperature of 20°C and a humidity of 80%-90% for 50 days, and then the fruiting bodies are harvested.

6. Use of the Cordyceps militaris embryo extract according to claim 3 for the preparation of a pesticide against tobacco mosaic virus, characterized in that, The specific steps of step (3) are as follows: the remaining culture residues are dried at 60°C, crushed, and sieved through an 80-mesh sieve to obtain Cordyceps militaris embryo powder.

7. Use of the extract of Cordyceps militaris embryo according to claim 3 for the preparation of a pesticide against tobacco mosaic virus, characterized in that, The specific steps of step (4) are as follows: the Cordyceps militaris embryo obtained in step (3) is mixed with ultrapure water at a mass-volume ratio of 1 g:80 mL for ultrasonic treatment, the power of ultrasonic treatment is 80 W, the temperature of ultrasonic treatment is 25°C, the time of ultrasonic treatment is 3 h, the solution is diluted with ultrapure water to 100 mL, and the supernatant is collected by centrifugation at 8000 r / min for 15 min to obtain the Cordyceps militaris embryo extract.

8. Use of the Cordyceps militaris embryo extract according to claim 3 for the preparation of a pesticide against tobacco mosaic virus, characterized in that, The dosage form of the tobacco mosaic virus-resistant pesticide is a liquid preparation, the liquid preparation is prepared by dissolving the Cordyceps militaris embryo extract in water, and the concentration of the Cordyceps militaris embryo extract in the tobacco mosaic virus-resistant pesticide is 0.01-0.05 g / mL.

9. Use of the extract of Cordyceps militaris embryo according to claim 3 for the preparation of a pesticide against tobacco mosaic virus, characterized in that, The concentration of the Cordyceps militaris embryo extract in the tobacco mosaic virus-resistant pesticide is 0.05 g / mL.

10. Use of the Cordyceps militaris embryo extract according to claim 3 for the preparation of a pesticide against tobacco mosaic virus, characterized in that, The application timing of the tobacco mosaic virus-resistant pesticide is the 5-6 leaf stage of tobacco, and the application amount is to form a transparent and uniform liquid film on the leaf surface without local accumulation and dripping after spraying.

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