Application of 4, 15-diacetyl verrucosporol in herbicide

Abstract

The invention belongs to the technical field of phytochemistry, and particularly relates to application of 4, 15-diacetyl verrucosporol in herbicides. The invention finds that the trichothecene compound 4, 15-diacetyl verrucosporol has herbicidal activity on main grassy weeds in a field, and has the potential of being 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. They can weed more accurately, improve crop yield and quality, reduce the impact on non-target organisms and the environment, and reduce pesticide residues and pollution. They can also address the problem of weed resistance, reduce weed control costs, help promote sustainable agricultural development, ensure food security, and promote the progress of green agriculture. Summary of the invention

[0005] In view of this, the present invention provides the use of 4,15-diacetyl verrucosporin in herbicides. The present invention discovers a trichothecene compound 4,15-diacetyl verrucosporin from a fungus, discloses a preparation method of the compound and its herbicidal activity against major grass weeds in the field, and the compound has the potential to be developed into a natural green biological herbicide.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

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

[0008] The invention uses silica gel, gel column chromatography, combined with recrystallization and high performance liquid chromatography technology to separate and purify a secondary metabolite from a highland barley pathogenic fungus Dactylobotrys graminicola to prepare a trichothecene compound; with the help of high-resolution mass spectrometry, nuclear magnetic resonance spectrum and X-ray single crystal diffraction, the structure of the trichothecene compound is identified and named as 4,15-diacetylverrucarol; the trichothecene compound 4,15-diacetylverrucarol has significant herbicidal activity against common grass weeds: Digitaria sanguinalis, Leptochloa chinensis, Japanese alopecuroides and Echinochloa crusgalli, and the molecular structure of the 4,15-diacetylverrucarol is as follows:

[0009] .

[0010] Further, the preparation method of 4,15-Diacetylverrucarol comprises the following specific steps:

[0011] (1) Bacterial fermentation

[0012] The strain Dactylobotrys graminicola DGC was inoculated into PSA (potato sucrose solid medium) for activation, and the activated strain was inoculated into rice medium for solid fermentation;

[0013] (2) Extraction and concentration

[0014] After step (1) is completed, the bacterial cells and the culture medium are placed in ethyl acetate for extraction, and the extract is concentrated under reduced pressure. The above process is repeated three times to obtain a crude extract.

[0015] (3) Separation and purification

[0016] The crude extract obtained in step (2) was dissolved in an organic solvent such as methanol or dichloromethane, mixed evenly with silica gel (200-300 mesh) at a mass ratio of 1:1.2, placed in a fume hood to dry naturally, and then crushed into powder. The mixed sample and column silica gel were loaded on the column using a dry method at a ratio of 1:20. The mobile phases of dichloromethane and methanol were 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32 and 0:100, respectively, to obtain 8 components Fr.1-Fr.8 with increasing polarity. Component Fr.2 was separated by a Sephadex LH-20 gel chromatography column, and then recrystallized to prepare the trichothecenes 4,15-Diacetylverrucarol.

[0017] In a second aspect, the present invention protects the use of the above-mentioned 4,15-diacetylverrucarol in the preparation of a herbicide.

[0018] In a specific embodiment, the herbicide further contains other relevant active ingredients for inhibiting the growth of weeds.

[0019] In a specific embodiment, the herbicide also contains relevant active ingredients such as safeners or growth regulators.

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

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

[0022] In a specific embodiment, the herbicide further contains other relevant active ingredients for inhibiting the growth of weeds.

[0023] In a specific embodiment, the herbicide also contains relevant active ingredients such as safeners or growth regulators.

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

[0025] In a fourth aspect, the present invention protects a method for controlling weeds, which is achieved by applying 4,15-diacetyl verrucosporidium or a herbicide containing 4,15-diacetyl verrucosporidium to an area where weeds grow.

[0026] In a specific embodiment, the grasses are primarily grass weeds.

[0027] In a more specific embodiment, the grass weeds are Digitaria sanguinalis, Leptochloa chinensis, Alopecuroides japonica, Echinochloa crusgalli and the like.

[0028] It can be seen from the above technical scheme that, compared with the prior art, the application of the trichothecene compounds disclosed in the present invention has the following beneficial effects: (1) by searching the SciFinder database, compared with the reported compounds of this type, the trichothecene compound 4,15-Diacetylverrucarol derived from the fungus of 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 separate and purify the target trichothecene compound 4,15-Diacetylverrucarol; (3) the trichothecene compound 4,15-Diacetylverrucarol has obvious inhibitory activity on the growth of grass weeds such as Digitaria sanguinalis, Leptochloa chinensis, Japanese Alopecuroides and Echinochloa crus-galli; and has no inhibition or influence on the growth of crops. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0033] Figure 4 The present invention shows the inhibitory effect of the compound 4,15-Diacetylverrucarol on the growth of Digitaria tangutica.

[0034] Figure 5 The present invention shows that the compound 4,15-Diacetylverrucarol has an inhibitory effect on the growth of barnyard grass.

[0035] Figure 6 The present invention shows that the compound 4,15-Diacetylverrucarol has an inhibitory effect on the growth of sweet intestine.

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

[0037] Figure 8 The compound 4,15-Diacetylverrucarol of the present invention has no inhibitory effect on the growth of crops (soybeans). DETAILED DESCRIPTION

[0038] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

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

[0040] (1) Bacteria activation and large-scale fermentation

[0041] The test strain Dactylobotrys graminicola DGC was inoculated on PSA (potato sucrose solid) medium and cultured in a constant temperature incubator at 25°C in the dark for 3 days. A hole was punched at the edge of the colony where the growth activity was best. 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 a constant temperature incubator at 25°C for 5-7 days.

[0042] Use a hole puncher to take a bacterial block from the edge of the activated strain, put it into the rice culture medium, and let it ferment in the dark at a constant temperature of 25°C for 12 days.

[0043] (2) Extraction, separation and purification

[0044] After the fermentation is completed, the bacteria and the culture medium are put into three times the volume of ethyl acetate for extraction, and the extract is concentrated under reduced pressure and the extraction and concentration are repeated three times to obtain a crude extract.

[0045] The 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 dry naturally, and then crushed into powder. The sample was loaded on the column using dry loading at a ratio of 1:20 with silica gel for column loading. The mobile phases of dichloromethane and methanol were 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32 and 0:100, respectively, to obtain 8 components Fr.1-Fr.8 with increasing polarity. Component Fr.2 (dichloromethane: methanol = 100:1) was separated by Sephadex LH-20 gel chromatography column, and then the trichothecenes 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 H NMR spectrum data is: 1 H NMR (CDCl 3 , 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 carbon spectrum data are: 13 C NMR (CDCl 3 , 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 spectrum of compound 4,15-Diacetylverrucarol is shown in Figure 2 The X-ray single crystal diffraction pattern of 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 Figure 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

Application of 1.4,15-diacetyl verrucosporin in inhibiting weed growth.

2. Application of 4,15-diacetyl verrucosporin in the preparation of herbicides.

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 a pesticide-acceptable carrier or adjuvant.

6. A herbicide, characterized in that: The herbicide contains 4,15-diacetyl verrucosporin.

7. The herbicide according to claim 6, characterized in that The herbicide also contains other relevant active ingredients for inhibiting the growth of weeds.

8. The herbicide according to claim 6, characterized in that The herbicide also contains a safener or a growth regulator.

9. The herbicide according to claim 6, characterized in that The herbicide also contains a pesticide-acceptable carrier or adjuvant.

10. A method for controlling weeds, characterized in that: This is accomplished by applying 4,15-diacetylthiocarboxol or a herbicide containing 4,15-diacetylthiocarboxol to the area where the weeds are growing.

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