Application of 4,15-diacetylverrucarol in herbicides

Abstract

The present invention belongs to the technical field of phytochemistry, and particularly relates to the application of 4,15-diacetylverrucarol in herbicides. The present invention discovers the herbicidal activity of the sesquiterpenoid compound 4,15-diacetylverrucarol against the main gramineous weeds in the field, and has the potential to be developed into a natural green biological selective herbicide.

Description

Technical Field

[0001] The invention belongs to the technical field of plant chemistry, and particularly relates to application of 4,15-diacetyl verrucosporin in herbicides. Background Art

[0002] Weeds pose a serious threat to agricultural production. They not only compete with crops for nutrients and light, but also serve as hosts for a variety of pests and diseases. Herbicides, as an important means of controlling weeds in modern agriculture, also face many problems and challenges. With the widespread use of herbicides, weeds have gradually evolved resistance, and this problem is becoming increasingly serious. The emergence of resistant weeds has greatly reduced the effectiveness of the originally highly effective herbicides, or even completely failed. The spread of resistant weeds not only increases the cost of weed control, but also forces farmers to use higher doses or more types of herbicides, thereby exacerbating environmental pollution and ecological damage. The development of herbicides with new mechanisms of action and targets is an effective way to solve this problem. Naturally derived compounds with herbicidal activity provide valuable resources for the development of new herbicides. Mycotoxins, as one of the important sources of herbicides, have unique mechanisms of action, selectivity, can accurately weed, protect crops, and are rich in fungal resources, which provides more possibilities for the development of new selective herbicides.

[0003] Selective herbicides refer to herbicides that can selectively kill or suppress certain weed species under certain dosages and conditions of use, while being relatively safe for crops or other target plants. This type of herbicide can specifically identify and act on weeds, accurately remove weeds in the field, reduce competition between weeds and crops for nutrients, water, light and other resources, ensure normal growth of crops, increase crop yield and quality, and is relatively safe for specific crops. While effectively preventing and controlling weeds, it will not cause significant damage to target crops, reducing the risk of weeding operations to crops. Herbicides such as Quizalofop-Ph-ethyl and Flupyralid-Ph-ethyl have good control effects on monocotyledonous weeds such as Crabgrass, Echinochloa crusgalli, and Alopecuroides in dicotyledonous crop fields. The selective mechanism of quizalofop-p-ethyl and fluazifop-p-ethyl is mainly reflected in the following aspects: First, in terms of absorption and conduction, the leaf morphology and structure of grass weeds are conducive to the absorption of the pesticide, and the vascular system enables the pesticide to be quickly transmitted to the growth point and other parts, while broad-leaved crops absorb less and transmit slowly; second, physiologically, the acetyl-CoA carboxylase of grass weeds is extremely sensitive to it, and the pesticide can inhibit the synthesis of fatty acids in weeds, while the enzyme of broad-leaved crops is not sensitive and has strong metabolic and detoxification capabilities, making them less vulnerable. However, long-term use of the same or the same type of selective herbicide can easily make weeds resistant to the pesticide, resulting in a decrease in the weed control effect, increasing the difficulty and cost of weed control, and may also have a toxic effect on non-target organisms such as beneficial insects and earthworms in farmland, destroying the ecological balance of farmland.

[0004] Therefore, the development of new selective herbicides is of great significance. It can weed more precisely, improve crop yield and quality, reduce the impact on non-target organisms and the environment, and reduce pesticide residues and pollution. It can also address the problem of weed resistance, reduce the cost of weeding, contribute to the sustainable development of agriculture, ensure food security, and promote the progress of green agriculture. Summary of the Invention

[0005] In view of this, the present invention provides the application of 4,15-diacetylverrucarol in herbicides. The present invention discovered a sesquiterpene compound 4,15-diacetylverrucarol from a strain of fungus, and disclosed the preparation method of this compound and its herbicidal activity against major gramineous weeds in the field. This compound has the potential to be developed into a natural green biological herbicide.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention protects the application of 4,15-diacetylverrucarol in inhibiting the growth of weeds.

[0008] The present invention uses silica gel, gel column chromatography, combined with recrystallization and high-performance liquid chromatography techniques to separate and purify the secondary metabolites of a strain of Dactylobotrys graminicola, a pathogenic fungus of highland barley, to obtain a sesquiterpene compound. With the help of high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy and X-ray single crystal diffraction, the structure of this sesquiterpene compound was identified and named 4,15-diacetylverrucarol; the sesquiterpene compound 4,15-Diacetylverrucarol has significant herbicidal activity against common gramineous weeds: Digitaria sanguinalis, Leptochloa chinensis, Alopecurus japonicus and Echinochloa crusgalli. The molecular structure of 4,15-diacetylverrucarol is as follows:

[0009] .

[0010] Furthermore, the preparation method of the 4,15-Diacetylverrucarol is as follows:

[0011] (1) Strain fermentation

[0012] Inoculate the strain Dactylobotrys graminicola DGC into PSA (potato sucrose solid medium) for activation, and then inoculate the activated strain into a rice medium for solid fermentation.

[0013] (2) Extraction and concentration

[0014] After step (1) is completed, the thallus and the culture medium are put into ethyl acetate for extraction, and the extract is concentrated under reduced pressure. After repeating the above process three times, a crude extract paste is obtained.

[0015] (3) Separation and purification

[0016] The crude extract obtained in step (2) is dissolved in organic solvents such as methanol and dichloromethane, mixed evenly with silica gel (200 - 300 mesh) in a ratio of 1:1.2 by mass, placed in a fume hood to air dry naturally, and then crushed into powder. Dry loading is carried out for column packing using the sample-mixing silica gel and the column-packing silica gel in a ratio of 1:20. Mobile phases with ratios of dichloromethane and methanol of 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32, and 0:100 are used respectively to obtain 8 fractions Fr.1 - Fr.8 with increasing polarity. Fraction Fr.2 is separated by a Sephadex LH-20 gel chromatography column, and then the trichothecene compound 4,15-Diacetylverrucarol is prepared by recrystallization.

[0017] In a second aspect, the present invention protects the application of the aforementioned 4,15-Diacetylverrucarol in the preparation of herbicides.

[0018] In a specific embodiment, the herbicide further contains other related active ingredients that inhibit weed growth.

[0019] In a specific embodiment, the herbicide further contains related active ingredients such as a safener or a growth regulator.

[0020] In a specific embodiment, the herbicide further contains a pesticide-acceptable carrier or excipient.

[0021] In a third aspect, the present invention protects a herbicide that contains the trichothecene compound 4,15-Diacetylverrucarol.

[0022] In a specific embodiment, the herbicide further contains other related active ingredients that inhibit weed growth.

[0023] In a specific embodiment, the herbicide further contains related active ingredients such as a safener or a growth regulator.

[0024] In a specific embodiment, the herbicide further contains a pesticide-acceptable carrier or excipient.

[0025] Fourthly, the present invention protects a method for controlling weeds, which is achieved by applying 4,15-diacetylverrucarol or a herbicide containing 4,15-diacetylverrucarol to the weed growth area.

[0026] In a specific embodiment, the weeds are mainly Gramineae weeds.

[0027] In a more specific embodiment, the Gramineae weeds are Digitaria sanguinalis, Leptochloa chinensis, Alopecurus japonicus, Echinochloa crusgalli, etc.

[0028] As can be seen from the above technical solutions, compared with the prior art, the application of the trichothecene compounds disclosed in the present invention has the following beneficial effects: (1) Through retrieval in the SciFinder database and comparison with the reported such compounds, the trichothecene compound 4,15-Diacetylverrucarol derived from fungi in the present invention is the first reported to have selective herbicidal activity; (2) The preparation method provided by the present invention can efficiently and accurately isolate and purify the target trichothecene compound 4,15-Diacetylverrucarol; (3) The trichothecene compound 4,15-Diacetylverrucarol has obvious inhibitory activity on the growth of Gramineae weeds such as Digitaria sanguinalis, Leptochloa chinensis, Alopecurus japonicus and Echinochloa crusgalli; it has no inhibitory effect on the growth of crops. Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art.

[0030] Figure 1 It is the molecular structure of the compound 4,15-Diacetylverrucarol of the present invention.

[0031] Figure 2 It is the high-resolution mass spectrum of the compound 4,15-Diacetylverrucarol of the present invention.

[0032] Figure 3 It is the X-ray diagram of the compound 4,15-Diacetylverrucarol of the present invention.

[0033] Figure 4 It is the inhibitory effect of the compound 4,15-Diacetylverrucarol of the present invention on the growth of Digitaria sanguinalis.

[0034] Figure 5 It is the inhibitory effect of the compound 4,15-Diacetylverrucarol of the present invention on the growth of Echinochloa crusgalli.

[0035] Figure 6 The inhibitory effect of the compound 4,15 - Diacetylverrucarol of the present invention on the growth of Amaranthus viridis.

[0036] Figure 7 The inhibitory effect of the compound 4,15 - Diacetylverrucarol of the present invention on the growth of Amaranthus retroflexus.

[0037] Figure 8 The compound 4,15 - Diacetylverrucarol of the present invention does not inhibit the growth of crops (soybeans). Detailed implementation manners

[0038] The present invention will be further described in detail below in conjunction with embodiments. Reagents or instrument and equipment not indicating the manufacturer are regarded as conventional products that can be purchased in the market.

[0039] Example 1: Specific process for the isolation and purification of 4,15 - Diacetylverrucarol

[0040] (1) Activation of the strain and large - scale fermentation

[0041] The test strain Dactylobotrys graminicola DGC was inoculated on PSA (potato sucrose solid) medium and cultured in the dark in an incubator at 25°C for 3 days. A puncher was used to punch at the place with the best growth activity at the edge of the colony, and the colony diameter was 6 mm. The colony was transferred and placed in the center of a new PSA solid medium plate and cultured in an incubator at 25°C for 5 - 7 days.

[0042] A puncher was used to take a mycelial block at the edge of the activated strain and put it into rice medium, and it was left to ferment statically in the dark at 25°C for 12 days.

[0043] (2) Extraction and isolation and purification

[0044] The whole fermented mycelium and medium were put into ethyl acetate with three times the volume for extraction. The extract was concentrated under reduced pressure and extracted and concentrated three times repeatedly to obtain a crude extract paste.

[0045] The obtained crude extract was dissolved in methanol, mixed evenly with silica gel (200 - 300 mesh) at a mass ratio of 1:1.2, placed in a fume hood to air dry naturally, and then crushed into powder. Column packing was carried out by dry loading with a ratio of sample mixing silica gel to column packing silica gel of 1:20. Mobile phases with ratios of dichloromethane and methanol of 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32, and 0:100 were used respectively to obtain 8 fractions Fr.1 - Fr.8 with increasing polarity. Fraction Fr.2 (dichloromethane:methanol = 100:1) was separated through a Sephadex LH - 20 gel chromatography column, and then the sesquiterpene compound 4,15 - Diacetylverrucarol was prepared by recrystallization.

[0046] (3)Compound structure identification

[0047] The molecular structure of the compound 4,15 - Diacetylverrucarol was determined by high - resolution mass spectrometry, one - dimensional nuclear magnetic resonance spectroscopy, and X - ray single - crystal diffraction ( Figure 1 ). The 1H NMR data are as follows: 1 H NMR (CDCl3, 500 MHz): δ 5.75 (dd, J = 7.9, 3.6 Hz, 1H), 5.45 – 5.41 (m, 1H), 4.16 (d, J = 12.3 Hz,1H), 4.04 (d, J = 12.3 Hz, 1H), 3.83 (d, J = 5.2 Hz, 1H), 3.74 (d, J = 5.6Hz, 1H), 3.13 (d, J = 4.1 Hz, 1H), 2.82 (d, J = 4.0 Hz, 1H), 2.53 (dd, J =15.5, 7.8 Hz, 1H), 1.71 (s, 4H), 0.80 (s, 3H). The 13C NMR data are as follows: 13 C NMR (CDCl3, 125MHz): δ C 170.9, 170.8, 141.0, 118.3, 79.1, 75.3, 66.8, 65.4, 63.6, 48.7, 48.0,43.1, 36.6, 28.0, 23.3, 21.2, 21.1, 6.7. The high - resolution mass spectrometry diagram of the compound 4,15 - Diacetylverrucarol is shown in Figure 2 . The X - ray single - crystal diffraction diagram of the compound 4,15 - Diacetylverrucarol is shown in Figure 3 .

[0048] Example 2: Growth inhibition of weeds and crops (soybean) by 4,15-Diacetylverrucarol

[0049] 4,15-Diacetylverrucarol was prepared into a 1 g / L stock solution with methanol, and diluted with clean water to 0.05, 0.1, and 0.2 g / L solutions for spraying. A clean water spray control was set, and each treatment was repeated 3 times.

[0050] Indoor whole-plant bioassay method was used. Plastic pots with a size of 7.0×7.0×7.8 cm were filled with nutrient soil (pH=6.3, organic matter content 1.1%) mixed with sand and nutrient matrix in a ratio of 2:1. The pots filled with soil were placed in plastic boxes and allowed to absorb water to saturation in advance. 20 weed seeds and 10 soybean seeds were evenly sprinkled in the water-absorbed pots. After sowing, a layer of fine soil was covered on the surface of the seeds. The pots and plastic boxes were placed in an indoor natural light incubator for cultivation. The cultivation temperature was 30 ℃ during the day and 25 ℃ at night. Thinning was carried out when the plants grew to the 3-leaf stage, and the number of plants per pot was set to 10. Two days later, the plants were sprayed on the stems and leaves using a 3WP-2000 walking spray tower (effective spray width 350 mm, spray height 300 mm, nozzle flow rate 390 mL / min, nozzle forward speed 291 mm / s). After the pesticide was dried, it was returned to the greenhouse for cultivation and watered 24 hours after the pesticide was applied. 21 days after the pesticide treatment, the above-ground parts of the plants were cut and weighed and recorded, and the fresh weight inhibition rate was calculated according to formula 1. SPSS software was used to calculate GR 10 , GR 90 , the selectivity index (Z) was calculated according to formula 2 to analyze the safety of the agent to soybeans. When Z < 2, it means it is unsafe to soybeans; when 2 < Z < 4, it means it is relatively safe to soybeans; when Z > 4, it means it is safe to soybeans.

[0051] Fresh weight inhibition rate (%) = (fresh weight of control group - fresh weight of treatment group) / fresh weight of control group × 100 —— Formula 1

[0052] Selectivity index (Z) = 10% soybean suppression dose / 90% weed suppression dose - Formula 2

[0053] Results Figures 4-8 And Tables 1-3.

[0054]

[0055]

[0056]

[0057] The results in Table 1 show that the compound 4,15-Diacetylverrucarol has a significant growth inhibitory effect on both Digitaria and Echinochloa crusgalli. Among them, at a concentration of 0.1 g / L, the fresh weight inhibition rates of Digitaria crusgalli and Echinochloa crusgalli were 92.87% and 91.41%, respectively, and the fresh weight inhibition rate of soybean was 3.24%. Visual observation after application showed that the fresh weight of soybean was not significantly different from that of the control group, and no obvious growth inhibition was observed.

[0058] As shown in Table 3, the selectivity index between soybean and weeds for stem and leaf treatment of 4,15-Diacetylverrucarol is 2.67, indicating that 4,15-Diacetylverrucarol is relatively safe for soybean. The results in Table 2 show that 4,15-Diacetylverrucarol has significantly different biological toxicity to Gramineae and broad-leaved weeds. 4,15-Diacetylverrucarol has good biological activity against Gramineae weeds, but poor inhibitory effect on broad-leaved weeds. It can hardly produce significant weed control effect at conventional dosages. Compared with Gramineae weeds, it requires several times or even ten times the toxin dosage to achieve the same control effect. However, in actual operation, high-dose medication is not economical and is prone to soil pollution, ecological imbalance and other problems, so it can be basically determined that the toxin is ineffective against broad-leaved weeds. Therefore, 4,15-Diacetylverrucarol has the potential to be a candidate target for herbicides for selective control of Gramineae weeds.

[0059] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. Application of 4,15-diacetyl verrucosporin in inhibiting the growth of weeds, wherein the weeds are crabgrass or barnyard grass.

2. Use of 4,15-diacetyl verrucosporin as an active ingredient in the preparation of a herbicide, wherein the grass is crabgrass or barnyard grass.

3. The use according to claim 2, characterized in that: The herbicide also contains other relevant active ingredients for inhibiting the growth of weeds.

4. The use according to claim 2, characterized in that: The herbicide also contains a safener or a growth regulator.

5. The use according to claim 2, characterized in that: The herbicide also contains pesticide-acceptable adjuvants.

6. A method for controlling weeds, characterized in that: This is accomplished by applying a herbicide containing 4,15-diacetylmyrtilbinol to an area where weeds are growing, the weeds being crabgrass or barnyardgrass.

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