CN114631546A

CN114631546A - Application of fleabane extract and herbicide

Info

Publication number: CN114631546A
Application number: CN202210334759.8A
Authority: CN
Inventors: 王趁; 王雨华
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-06-17
Anticipated expiration: 2042-03-31
Also published as: CN114631546B

Abstract

The invention relates to an application of a fleabane extract and a herbicide, and relates to the technical field of herbicides. The main technical scheme adopted is as follows: use of a flea-grass extract as, or in the preparation of, a herbicide for the removal of Bidens pilosa; the invention mainly provides a new application of flea-grass extract in serving as or preparing a bidens bipinnata herbicide.

Description

Application of fleabane extract and herbicide

Technical Field

The invention relates to the technical field of herbicides, in particular to an application of a flea grass extract and a herbicide.

Background

Weeds are one of the major biological factors responsible for crop yield loss. From the time of original farming to modern agricultural systems today, effective control of weeds as an important component of agricultural ecosystems has been a problem worldwide.

With the completion of 2, 4-dichlorophenoxyacetic acid, the first artificial herbicide in the 40 th of the 20 th century, the chemical synthetic herbicide gradually replaced the traditional weeding method and became the main mode for controlling weeds in agricultural systems. The arrival of the artificially synthesized herbicide frees the labor force under the traditional weeding mode and ensures the yield of crops; at the same time, however, the large-scale application of herbicides poses a series of problems such as pesticide residues and weed resistance, and the threat to human and environmental health caused thereby has raised worldwide attention. Scientists around the world have thus turned their attention to alternative methods for weed control based on natural products.

Among natural products, the potential of essential oils for use in agriculture has received great attention because of their broad range of biological effects, particularly as sources of natural pesticides, natural bactericides and insecticides. The essential oil not only has insecticidal and antibacterial activities, but also has strong phytotoxicity potential due to the structural diversity and composition diversity of the compounds, and shows strong biological activity on weed seed germination and seedling growth. Over the past two decades, many researchers have verified the phytotoxicity of essential oils to weeds, and have explored the potential of essential oils in controlling weeds. At present, the herbicides with essential oil as the main component are mainly from aromatic plant essential oils (Shaffer 2019; Shiffle 2019) such as clove (Syzygium aromaticum), Clinopodium polycephalum (Satureja hordens), Cinnamomum zeylanicum (Cinnamomum zeylanicum I), Thymus sieboldii (Thymus zygium) and Cymbopogon citratus) in the American market. However, synthetic herbicides are the main way of controlling weeds in agricultural production in china to date, and natural herbicides derived from plant essential oils have not been found in the market in china. In view of the above, there is a great need to study herbicides of natural origin for use in agricultural production.

Disclosure of Invention

In view of the above, the present invention provides an application of a flea-grass extract and a herbicide, and mainly aims to provide a new application of the flea-grass extract in the preparation of a bidens bipinnata herbicide.

In order to achieve the purpose, the invention mainly provides the following technical scheme:

in one aspect, the present invention provides the use of a flea grass extract as, or in the preparation of, a herbicide for the removal of Bidens pilosa.

Preferably, the flea grass extract is flea grass essential oil.

Preferably, the essential oil of fleabane comprises: alpha-terpinene, p-cymene, gamma-terpinene, terpinen-4-ol, carvacrol methyl ether, carvacrol, beta-bisabolene and moschus ether A.

Preferably, in the essential oil of flea grass: the content of the alpha-terpinene is 1-7%; the content of p-cymene is 3-18.5%; the content of the gamma-terpinene is 18.0-40.3%; the content of terpinen-4-ol is 0.05-0.6%; the content of carvacrol methyl ether is 3-18%; the content of carvacrol is 12-38%; the content of the beta-bisabolene is 0.7-12%; the moschus ether A content is 0.02-0.1%.

Preferably, the flea-killing essential oil also contains alpha-arborvitae, alpha-pinene, beta-pinene, myrcene, alpha-phellandrene, 3-carene, limonene, cis-ocimene, isoterpinene, fenchyl ketone, linalool, borneol, alpha-terpineol, thymol, trans-caryophyllene, farnesene, lupinene, beta-sesquiphellandrene and caryophyllene II oxide.

Preferably, in the essential oil of flea grass: the content of the alpha-arborvitae is 1.4-2.3%; the content of the alpha-pinene is 0.1-0.45%; the content of the beta-pinene is 0.13-0.15%; the content of myrcene is 0.08-0.18%; the content of the alpha-phellandrene is 0.07-0.155%; the content of the 3-carene is 1.5-2.4%; the content of the limonene is 1.9-3.5%; the content of the cis-ocimene is 0.08-0.16%; the content of the terpinolene is 0.2-2.3%; the content of the fenchone is 0.1-0.42%; the linalool content is 0.02-0.095%; the content of the borneol is 0.07-0.45%; the content of the alpha-terpineol is 0.05-0.19%; the content of thymol is 0.45-0.75%; the content of the trans-caryophyllene is 0.35-0.67%; the content of the farnesene is 0.04-0.099%; the content of the lupinene is 0.4-1.993%; the content of the beta-sesquioenanthe is 0.15-0.291%, and the content of the caryophyllene oxide II is 0.120-0.29%.

Preferably, the preparation method of the fleabane essential oil comprises the following steps: extracting oil from the flea grass by adopting essential oil distillation equipment to obtain the flea grass essential oil.

In another aspect, embodiments of the present invention provide a herbicide, wherein the herbicide comprises a flea grass extract.

Preferably, the flea grass extract is flea grass essential oil.

Preferably, the concentration of the essential oil of flea grass in the herbicide is 5 mul/L to 30 ml/L.

Compared with the prior art, the application and the herbicide of the flea grass extract have at least the following beneficial effects:

in one aspect, the present embodiments provide for the first time a new use of a flea-grass extract, i.e., a flea-grass extract for use as or in the preparation of a herbicide for the removal of Bidens pilosa. Here, the flea-grass extract is preferably flea-grass essential oil, which has an excellent herbicidal effect on bidens bipinnata.

Further, the embodiments of the present invention find that the flea grass essential oil has a synergistic effect (mainly the synergistic effect of alpha-terpinene, p-cymene, gamma-terpinene, terpinen-4-ol, carvacrol methyl ether, carvacrol, beta-bisabolene, moschus ether a) among various components, so that the flea grass essential oil has an excellent effect of removing sticktight.

In another aspect, embodiments of the present invention also provide a herbicide, wherein the herbicide comprises a flea grass extract (preferably flea grass essential oil); the herbicide is mainly used for removing the sticktight.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a TIC chart of the chemical composition of essential oil of Siphonaena;

FIGS. 2A-2C are schematic illustrations of the effect of essential oil of flea grass on the germination of Bidens pilosa seeds and the growth of seedlings; wherein, fig. 2A is a schematic diagram of the effect of solutions of essential oil of zoysia pulchera on the germination rate of seeds at different concentrations; FIG. 2B is a graph showing the effect of various concentrations of essential oil solutions of flea grass on shoot growth; FIG. 2C is a graph showing the effect of various concentrations of essential oil solutions of Dolichos pulcherus on gibberellin 3 content;

FIG. 3 is a graph showing the disturbance of the endogenous hormone content of seedlings of Bidens pilosa at day 7 of seed germination under the treatment of a solution of essential oil of Bidens pilosa.

FIG. 4 is a graph of the growth of Bidens pilosa plants 25 days after foliar spray treatment of a solution of essential oil of flea grass;

FIG. 5 is a schematic illustration of the effect of foliar application of a solution of essential oil of flea grass on the endogenous hormone content of Bidens pilosa leaves; wherein, the graph (a) is a schematic diagram of the effect of solutions of essential oil of fleabane in different concentrations on the auxin content; panel (b) shows the concentration of the essential oil solution of flea grass on GA₃The influence of the content is shown in the figure; FIG. (c) is a graph showing the effect of various concentrations of essential oil solutions of flea grass on jasmonic acid content; FIG. d is a graph showing the effect of various concentrations of essential oil solutions of Siphonium parvifolium on the amount of isoleucine jasmonate; FIG. (e) is a graph showing the effect of various concentrations of essential oil solutions of flea grass on salicylic acid content; FIG. (f) is a graph showing the effect of various concentrations of essential oil solutions of flea grass on abscisic acid content;

FIG. 6 is a graph showing the effect of foliar application of a solution of essential oil of Fasciola on the photosynthetic pigment content and photosynthetic rate of Bidens bipinnata plants; FIG. (a) is a graph showing the effect of various concentrations of essential oil solutions of flea grass on chlorophyll a content; graph (b) shows the concentration of the essential oil solution of fleabane on the leaf greenSchematic of the effect of the content of element b; FIG. (c) is a graph showing the effect of various concentrations of essential oil solutions of flea grass on carotenoid content; graph (d) shows the maximum conversion efficiency (F) of solutions of essential oil of Douglas grass to light System II_v/F_M) Schematic diagram of the effect of (c).

FIG. 7 is a schematic illustration of the effect of foliar application of a solution of essential oil of flea grass on the activity of the antioxidant enzymes and the malondialdehyde content of Bidens pilosa leaves; FIG. (a) is a graph showing the effect of various concentrations of essential oil solutions of fleabane on SOD activity; FIG. (b) is a graph showing the effect of various concentrations of essential oil solutions of flea grass on CAT activity; FIG. (c) is a graph showing the effect of various concentrations of an essential oil solution of zoysia on POD activity; FIG. (d) is a graph showing the effect of various concentrations of essential oil solutions of fleabane on APX activity; panel (e) is a graphical representation of the effect of various concentrations of essential oil solutions of zoysia on MDA content.

Detailed Description

To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description of the embodiments, structures, characteristics and effects according to the present invention will be made with reference to the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Flea grass (Adenosma brachiosum Bonatai) is an annual aromatic herb of the Plantaginaceae, which is intercropped in upland and or corn fields by the local Hani nationality. The flea grass is used as a traditional herbal medicine by local people to treat various diseases such as cold, skin itch and the like, and the essential oil of the flea grass also has the effect of repelling mosquitoes. With the increase of the demand of the market for the essential oil of the flea grass, local farmers start to plant a large amount of the flea grass in a mode of intercropping between crops such as dry rice, corn and the like, sowing before the rainy season comes, and harvesting in the full-bloom stage of 10-11 months.

Bidens pilosa L is a common malignant weed in local farmlands and roadside, has strong growth advantage, and interferes and limits the growth of crops.

The invention mainly researches the allelopathy of local plant flea grass to local weed Bidens bipinnata. In addition, researches in the prior art find that the plant essential oil can cause oxidative damage by directly acting on a cell membrane layer, and reduce the content of photosynthetic pigments, so that the seed germination and seedling growth are interfered, and the weed seed germination and seedling growth are inhibited. Therefore, the invention designs seed germination and potted seedling growth experiments, and determines the influence of the essential oil of the flea grass on the germination of the bidens bipinnata seeds and the growth of the seedlings by determining the photosynthetic pigment, the activity of the antioxidant enzyme and the content of the hormone.

The invention is illustrated below by means of specific experimental examples:

the main instruments used in the present invention are set forth in table 1:

TABLE 1

Name of instrument	Type number	Manufacturer of the product
			Essential oil distillation equipment	RY–JYTQ–200L	Shanghai Ruiyuan machinery and Equipment Co Ltd
Gas chromatography-mass spectrometer	Agilent 7890A GC-5975C MS	Agilent technologies, Inc. of USA
			Chromatographic column	HP–5:50m×0.2mm×0.33μm	Agilent technologies, Inc. of USA
Illumination incubator	GXZ–310D	Ningbo Southeast Instrument Co.,Ltd.
			Professional root system analysis system	WinRHIZOTM，2013e	Regent Instruments, Canada Inc
Liquid chromatogram-mass spectrum combined instrument	Shimadzu LCMS–8040	Shimadzu Japan Ltd
			Centrifugal machine	Eppendorf 5804R	Germany Albende stockings Ltd
Enzyme linked immunoassay analyzer	Tecan Infinite M200 pro	Tecan of Switzerland
			Ultraviolet spectrophotometer	Thermo Scientific BioMate 3S	THERMO FISHER SCIENTIFIC Inc.
Modulated chlorophyll fluorescence imager	WALZ Imaging–Pam 2000	Germany WALZ Co
			Vacuum centrifugal concentrator	Eppendorf Concentrator plus	Germany Albende stockings Ltd
Full-automatic sample rapid grinding instrument	Tissuelyser–24	Shanghai Jingxin science & technology Ltd
			Ultrapure water preparation system	Heal Force SMART–M	SHANGHAI CANREX ANALYSIS INSTRUMENT Co.,Ltd.
Full-automatic snow ice maker	XUEKE，IMS–20	CHANGSHU XUEKE ELECTRIC Co.,Ltd.
			Electronic analytical balance	Sartorius BSA224S–CW	Seidoris, Germany
Super clean bench	SW–CJ–1D	Shanghai Jing medical instruments Co Ltd
			Digital display three-temperature water bath pot	DK–8A	Changzhou Gauder instruments manufacturing Co Ltd
PH meter	Thunder magnet PHS-3E	SHANGHAI INESA SCIENTIFIC INSTRUMENT Co.,Ltd.
			Ultrasonic cleaning machine	nissei KD–300	Japan Fengcheng Power machine Co Ltd

The main reagent consumables used by the flea grass essential oil for the growth and allelopathy of the sticktight are shown in the table 2:

TABLE 2

Example 1

Example 1 a flea grass essential oil was mainly extracted as follows:

1. experimental materials:

fresh plants of flea grass are collected from the flea grass planting base in Meng La county of Xishuangbanna, Yunnan province. In the afternoon of sunny weather, reaping the overground part of the plant in full-bloom period by a sickle, removing weeds, bundling into small bundles, and harvesting the oil in the same day.

Bidens pilosa seeds were collected from the flea grass planting field. Collecting the collected seeds in a paper collection bag, placing in a cool and ventilated place, drying in the shade, removing impurities and unsaturated seeds, and storing in a refrigerator at 4 ℃.

2. Experimental methods

2.1 extracting essential oil of fleabane:

(1) putting the sample into a basket: 20 kg of samples are loaded once (calculated by fresh samples), the samples are transversely cut for 2 to 3 times by a hand hay cutter and are vertically loaded into a basket;

(2) putting a distillation basket into a tank: hanging the distillation basket into the tank, and screwing down the tank cover;

(3) steam heating: firstly, a power switch in the power box is turned on, then a switch of the steam generator is turned on, and the steam generator is switched from a low gear to a high gear;

(4) an air supply switch: observing the pressure gauge, opening the air supply switch to supply air to the tank when the pointer reaches 0.2, and observing the temperature on the temperature screen of the tank by hearing the silk sound of the air supplied to the tank;

(5) opening circulating water: when the temperature in the tank rises to 75 ℃, a water pump power supply and a fan power supply in the power box are sequentially turned on;

(6) and (3) distillation: distilling over water for 6 h;

(7) oil receiving: measuring the oil column with a ruler, collecting the upper layer volatile oil with a measuring cylinder when the height of the oil column reaches 9cm, adding anhydrous sodium sulfate, drying, placing in a brown bottle, storing in a refrigerator at 4 deg.C, and performing subsequent essential oil component analysis and bioactivity determination. The obtained fleabane essential oil is light yellow, and the yield of the fleabane fresh plant essential oil is 0.7% (w/v).

2.2: GC-MS analysis condition and identification method of chemical components of fleabane essential oil

(1) GC-MS analysis conditions

GC conditions were as follows: agilent HP-5 column (30 m. times.0.25 mm. times.0.25 μm); the temperature raising program is that the initial temperature is 50 ℃, and the temperature is kept for 1 min; raising the temperature to 250 ℃ at the speed of 5 ℃/min, and keeping the temperature for 5 min; then increased to 300 ℃ at a rate of 10 ℃/min. The total run time was 50 min. The carrier gas is high-purity helium (99.999%) with the flow rate of 2.1 mL/min; the injection port temperature is 250 ℃; the split ratio is 20: 1; the amount of sample was 1.0. mu.L.

MS conditions: the temperature of a chromatography-mass spectrometry interface is 280 ℃, the ionization mode EI is adopted, the electron bombardment energy is 70eV, and the scanning range is 50-550 m/z; the ion source temperature is 230 ℃; the temperature of the quadrupole mass analyzer is 150 ℃; the ionization mode is electron impact. MS database: NIST17 mass spectrometry database (Adams 2007).

(2) Qualitative analysis of essential oil composition

The chemical components of the essential oil of the fleabane are mainly identified by the following modes: comparing the mass spectrum fragments of the essential oil components with the existing spectrograms in a spectrum library on the basis of a NIST17 mass spectrum database, wherein the Match value is more than or equal to 900 and is used as a candidate compound; calculating retention indexes RICal of all essential oil components by using the n-alkanes of the homologous series, and comparing the retention indexes RICal with retention indexes (RILit) of compounds in literature reports to determine target compounds; and thirdly, performing comparison on main chemical components in the essential oil of the flea grass by using a standard substance to finally determine the compound.

Through the component analysis, the chemical components of the fleabane essential oil are as follows: see fig. 1 and table 3. Wherein, FIG. 1 is a TIC chart of chemical components of essential oil of flea grass; the numbers of chromatographic peaks in FIG. 1 correspond to the numbers of the compounds in Table 3.

The yield of the essential oil of the overground part of the fresh plant of the flea grass is 0.07 percent. As shown in figure 1 and Table 3, 27 chemical components of the essential oil of Siphonria dubia, which accounts for 98.892% of the total components of the essential oil, were identified together by GC-MS analysis. Wherein, the gamma-terpinene (34.927%), the carvacrol (14.033%), the carvacrol methyl ether (12.542%), the paracymene (10.046%) and the beta-bisabolene (8.199%) are main components of the essential oil of the flea-flea, and the total proportion of the essential oil accounts for 79% of the total amount of the essential oil of the flea-flea. The chemical component types of the fleabane essential oil are mainly hydrocarbon monoterpene (59.153%), oxidized monoterpene (28.378%) and hydrocarbon sesquiterpene (11.238%).

TABLE 3

Here, it should be noted that: the flea-killing essential oil contains 8 components (the 8 components comprise alpha-terpinene, p-cymene, gamma-terpinene, terpinen-4-ol, carvacrol methyl ether, carvacrol, beta-bisabolene and moschus ether A) which are functional components for removing sticktight from flea.

In addition, it should be noted that: essential oil extraction was carried out on flea grass grown under different conditions (different regions, different seasons, different batches) and it was found that the content of these 8 components varies, substantially within the following ranges: the content of alpha-terpinene is 1-7%; the content of p-cymene is 3-18.5%; the content of gamma-terpinene is 18.0-40.3%; the content of terpinen-4-ol 4 is 0.05-0.6%; the content of carvacrol methyl ether is 3-18%; the content of carvacrol is 12-38%; the content of beta-bisabolene is 0.7-12%; the moschus ether A content is 0.02-0.1%.

For flea grass growing under different conditions (different regions, different seasons, different batches), the extraction of essential oil was carried out, except for the above 8 ingredients: the content of the alpha-arborvitae is 1.4-2.3%; the content of the alpha-pinene is 0.1-0.45%; the content of the beta-pinene is 0.13-0.15%; the content of myrcene is 0.08-0.18%; the content of the alpha-phellandrene is 0.07-0.155 percent; the content of the 3-carene is 1.5-2.4%; the content of the limonene is 1.9-3.5%; the content of the cis-ocimene is 0.08-0.16%; the content of the terpinolene is 0.2-2.3%; the content of the fenchone is 0.1-0.42%; the linalool content is 0.02-0.095%; the content of the borneol is 0.07-0.45%; the content of the alpha-terpineol is 0.05-0.19%; the content of thymol is 0.45-0.75%; the content of the trans-caryophyllene is 0.35-0.67%; the content of the farnesene is 0.04-0.099%; the content of the lupinene is 0.4-1.993%; the content of the beta-sesquioenanthe is 0.15-0.291%, and the content of the caryophyllene oxide II is 0.120-0.29%.

Example 2

Example 2 extraction of essentially one essential oil of flea grass; among them, example 2 differs from example 1 in that the fresh plants of flea grass used in example 2 were taken from the Xishuangbanna tea garden.

See example 1 for extraction methods.

See example 1 for compositional analysis methods.

The chemical composition of essential oil of flea grass of this example is shown in table 4:

TABLE 4

Example 3

Example 3 extraction of essentially one essential oil of flea grass; among them, example 3 is different from example 1 in that the fresh plants of flea grass used in example 3 were collected from the Kunming plant research institute resource plant cultivation greenhouse of Chinese academy of sciences.

See example 1 for extraction methods.

See example 1 for compositional analysis methods.

The chemical composition of essential oil of fleabane of this example is shown in table 5:

TABLE 5

The daphnia essential oil provided by the embodiment of the invention is used for a chemical sensation experiment on the growth of Bidens pilosa:

1. the flea grass essential oil prepared in the example 1 is prepared into a flea grass essential oil solution, and the preparation method comprises the following steps: taking 2.5ml of flea grass essential oil, adding Tween80 (1%, v/v) for dissolving, adding 400ml of double distilled water for fully dissolving, performing ultrasonic treatment for 30min, taking out every 10min, and shaking up once to prepare the essential oil solution mother liquor with the concentration of 1 ml/L. Then diluted into essential oil solution with the concentration of 50 mul/L, 100 mul/L, 250 mul/L, 500 mul/L and 1000 mul/L by double distilled water (1% Tween80, v/v) for seed germination experiments. With reference to the above method, the essential oil solutions with diluted volume concentrations of 0.5%, 1.0%, 2.5%, 5.0% and 10% of the essential oil mother liquor were used for foliar spray experiments.

2. Bidens pilosa seed germination experiment

The experiment adopts a culture dish experiment method, and the operation is as follows: (1) taking a plurality of plump bidens bipinnata seeds with uniform shape, putting the seeds into a 50mL centrifuge tube, soaking and sterilizing the seeds for 5min by using a 5% volume sodium hypochlorite (NaClo) aqueous solution, washing the seeds for 5 times by using sterile water, and placing the seeds on filter paper to be dried for later use. (2) A culture dish with the diameter of 9cm is taken, 2 layers of filter paper are paved, 5mL of essential oil solution is added, 20 seeds are uniformly sowed on the filter paper, and the filter paper is sealed by a Parafilm sealing film. Each treatment was set to 5 replicates. (3) Randomly placing the culture dishes in an illumination incubator, wherein the parameters of the incubator are set to 3000LX of illumination intensity, the illumination time is 12h, and the temperature is 30 ℃; dark 12h, temperature 25 ℃. The culture is carried out for 7 days, and the germination quantity of the seeds is observed and recorded every 24 hours. After culturing for 7 days, the germination rate of the bidens bipinnata seeds, the lengths of the roots and stems of the seedlings and the content of endogenous hormones under different concentration treatment are respectively counted. The whole experiment was repeated 3 times.

Bidens pilosa seed Germination Rate (GP)

The standard of seed germination is that the breakthrough length of the radicle of the seed is more than or equal to 1 mm. The calculation formula of the seed germination rate is as follows:

in the formula, SNG is the number of germinated seeds, and SN0 is the number of single culture dishes.

Determination of root and stem length of young spanishneedles herb

The length of the root and stem of the seedling is measured by adopting a WinRHIZOTM root system analysis system. The specific operation method is referred to (Wang & Zhang 2009), and the specific system parameters of winrhiztm in the experiment are set as follows, and the mode is as follows: a professional mode; the type of the manuscript is as follows: film (with film area guide); film type: correcting; image type: 8-bit gray scale; resolution ratio: 600 dpi; size of manuscript: 203 x 254 mm; target size: an original document; a regulator: and USM sharpening.

The experimental results are as follows:

the results of the germination experiments of the bidens bipinnata seeds treated by the solutions of the flea grass essential oil with different concentrations are shown in figures 2A-2C: the germination rate of the bidens bipinnata seeds is reduced along with the increase of the concentration of the treatment solution; the inhibitory effect of flea-grass essential oil on the germination of Bidens pilosa seeds decays with time. When the treatment concentration was higher (500. mu.l/L, 1000. mu.l/L), the germination of the seeds was completely inhibited. At the lowest concentration treatment (50. mu.l/L), the effect of the essential oil solution on seed germination was small, showing only a weak effect at day 7 (see FIG. 2A). For the same concentration treatment, the inhibition effect of all concentration treatments on the germination of the bidens bipinnata seeds is reduced along with the prolonging of the germination duration, wherein the medium-high concentration treatment always shows stronger inhibition effect.

In germination experiments, treatment of the flea grass essential oil with different concentrations also has obvious influence on the growth of the sticktight seedlings. Similar to the seed germination rate, the medium-high concentration has obvious inhibition effect on the root and stem length of the bidens bipinnata, while the low-concentration essential oil solution treatment has weaker inhibition effect on the root and stem growth of the bidens bipinnata, and the flea grass essential oil has stronger inhibition effect on the stem growth than the root (see figure 2B). Compared with the control group, the medium-high concentration treatment (250. mu.l/L, 500. mu.l/L and 1000. mu.l/L) has obvious inhibition effect on the growth of roots and stems of the bidens bipinnata, and the root and stem lengths of the seedlings of the bidens bipinnata are not obviously different from those of the control group under the lowest concentration treatment (50. mu.l/L).

To further explore the mechanism by which essential oil of flea grass inhibits the germination of Bidens pilosa seeds, we measured the content of the endogenous hormones auxin (IAA), Jasmonic Acid (JA), Jasmonic acid isoleucine (JA-Ile), Salicylic Acid (SA), and Abscisic acid (ABA) in Bidens pilosa seedlings at day 7 of germination. Analysis on the results of endogenous hormone content shows that the treatment of essential oil solutions with different concentrations affects GA in the sticktight seedlings₃(gibberellac acid 3, gibberellin 3) content (fig. 2C), and decreased with increasing concentration of the essential oil solution. GA of Bidens pilosa seedlings compared to the control group₃The content is obviously increased under the treatment of low-concentration essential oil solution and is obviously reduced under the treatment of medium-high concentration. Bidens pilosa seedlings GA at low concentration treatment (50, 100. mu.l/L)₃Compared with a control group, the content is respectively increased by 119.47 and 54.75 percent; when the concentration of essential oil is increased to (250, 500, 1000. mu.l/L), the seedling GA of Bidens pilosa is obtained₃The content of (b) is respectively reduced by 45.13,53.98, and 54.75%. However, the contents of IAA, JA-Ile, SA, ABA and other endogenous hormones in the Bidens pilosa seedlings are changed, but the contents of the endogenous hormones show high and low irregular changes along with the increase of the treatment concentration of the essential oil solution (see figure 3).

3. Bidens pilosa seedling growth experiment

In order to determine the influence of the foliar spraying of the daphnia fop-butyl essential oil solution on the growth and development of the sticktight seedlings, an indoor pot culture experiment was carried out in 2021 for 3-9 months. For the experiment, a plastic flowerpot (with an upper caliber of 16cm, a lower caliber of 12cm and a height of 11.5cm) with a volume of 2L is filled with 1.5kg of soil (75% of Pindstrup substrate and 25% of perlite). Sowing 20 seeds of the bidens bipinnata in each pot, thinning the seedlings when the seedlings come out of the soil and grow to 2 complete true leaves, and only keeping 5 plants with uniform growth vigor and consistent size in each pot. When all the plants grow to the 4-6 leaf stage, carrying out a leaf surface spraying experiment of the essential oil solution of the flea grass.

The flea-flea essential oil solution is prepared in the same way, namely, mother liquor with the concentration of 1ml/L is prepared firstly, and then the mother liquor is diluted by using 1 percent Tween 80-containing aqueous solution.

Reference to the foliar spray method (Pouresmaeil et al.2020), a manual brown watering can (100 ml/m) was used²) Spraying flea grass essential oil solutions with different concentrations on the surfaces of the bidens bipinnata, wherein the spraying amount of the leaves is 125mg/m respectively²、25mg/m²、625mg/m²、1250mg/m²And 2500mg/m². The leaf surface spraying experiment is carried out between 9 and 11 noon, and the spraying is carried out once every 5 days, the treatment period is 20 days, and the spraying is carried out for 5 times in total. Each treatment concentration was set to 5 replicates and the entire potting experiment was repeated 3 times. After the foliage spraying treatment of the flea grass essential oil solution is finished, sampling, and respectively measuring the endogenous hormone content, the chlorophyll content, the photosynthetic rate, the antioxidase activity and the malondialdehyde content of the leaves.

The experimental results are as follows:

in greenhouse pot experiments, compared with a control group, after the daphnia grass essential oil solution is subjected to foliage spraying treatment for 20 days, the growth state and appearance form of the sticktight plant are obviously changed, and the plant grows along with the increase of the treatment concentration of the essential oil solutionThe greater the inhibition of growth, the greater the morphological differences of the aerial parts of the plants (see FIG. 4). Compared with the control group, the Bidens bipinnata after the treatment of different concentrations is characterized in that the page of the low-concentration essential oil solution is sprayed and treated for 25 days (less than or equal to 250 mg/m)²) The growth vigor of the bidens bipinnata plants is weak, no lateral bud exists, leaves are yellow, partial plants are changed from vegetative growth to reproductive growth, and buds appear; as the concentration of the treatment solution increased (625 mg/m)²，1250mg/m²，2500mg/m²) The stress on the plant is enhanced, the plant is short and small, no lateral bud exists, the growth of the terminal bud is inhibited, and the edge of the leaf is curled, browned to dry; when the treatment concentration reaches 2500mg/m²At that time, some plants stop growing or even die.

4. Pot-carried plant leaf hormone sample collection and content determination

In the greenhouse experiment, 1h after 5 th foliar spray treatment, the leaves of the Bidens pilosa are sampled to investigate the response of endogenous hormones in the Bidens pilosa leaves under the foliar spray treatment of solutions of flea grass with different concentrations.

The experimental results are as follows:

plant essential oils contain an abundance of allelochemicals which have inhibitory effects on weed seed germination and seedling growth (Dudai et al 1999; Angelini et al 2003; El Sawi et al 2019; Verdeguer et al 2020). Plant endogenous hormones play an important regulatory role during plant growth, especially during plant-to-plant interactions, where they play a key role in the response to allelochemicals (Cheng)&Cheng 2015). Therefore, we determined the content of IAA, GA, JA-Ile, SA and ABA in Bidens bipinnata leaves under different concentrations of treatment with the essential oil solution of the flea grass. Analysis of the measurement result of the content of the endogenous hormone shows that the content of growth related hormones IAA and GA is reduced along with the increase of the concentration of the treatment solution; while the contents of growth stress hormones JA, JA-Ile, SA and ABA were increased (FIG. 5). But the sensitivity of different hormones to the treatment solution varies. At the highest concentration treatment (2500 mg/m)²) Auxin IAA and GA₃The content of (A) decreased by 52.43 and 68.05%, respectively (FIGS. 5 a-b); JA. Containing JA-Ile, SA and ABAThe amount was increased by 31, 108.7, 5.26 and 2 fold, respectively, compared to the control group (fig. 5 c-f).

5. Determination of content of photosynthetic pigment and photosynthetic rate of bidens bipinnata

(1) Chlorophyll extraction and content determination

The specific operation method of the experiment is as follows by combining the biological characteristics of the bidens bipinnata: about 200mg of fresh leaves are taken and put into a 2mL centrifuge tube, and the centrifuge tube is quickly put into liquid nitrogen for freezing storage. Grinding into powder with a cryo-grinder, adding 1mL of 85% propanol solution, and extracting for 30min on ice in the dark. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min. And (3) taking 200 mu L of the supernatant to a 96-well plate, respectively measuring the light absorption values under the wavelengths of 663nm, 645nm and 440nm by using an enzyme-linked immunoassay analyzer, and respectively calculating the concentrations of chlorophyll and carotenoid according to the following formulas.

Chlorophyll a concentration (mg/L) Chlorophyl a ═ 12.7A₆₆₃-2.69A₆₄₅

Chlorophyll b concentration (mg/L) Chlorophyl b ═ 22.9A₆₄₅-4.68A₆₆₃

Carotenoid concentration (mg/L) ═ 4.7A₄₄₀-(1.38A₆₆₃+5.48A₆₄₅)

(2) Determination of herba Bidentis Bipinnatae photosynthetic efficiency under dark adaptation

The modulated chlorophyll fluorescence imager (Imaging-Pam 2000) can collect chlorophyll fluorescence images of plants by measuring distribution information of chlorophyll fluorescence signals in a two-dimensional space, and detect the time-space heterogeneity of plant photosynthesis by combining a digital image processing technology. And detecting the change of the photosynthesis of the bidens bipinnata leaves under the condition that the bidens bipinnata essential oil with different concentrations is sprayed and treated by using a modulated chlorophyll fluorescence imager. The specific operation is as follows: after the foliar spray treatment was completed, different groups of sticktight were dark-adapted for 30min, placed in a measuring box, and the initial fluorescence F0 was measured, followed by saturation pulsing (6000. mu. mol)^-2s^-1) Treatment is carried out for 0.8s, and the maximum fluorescence Fm under dark adaptation is measured. The conversion efficiency of the photosynthesis of the bidens bipinnata is calculated according to the formula Fv/Fm ═ Fm-F0)/Fm.

The experimental results are as follows:

the photosynthetic pigment content and photosynthetic rate are shown in FIG. 6. Dissolving herba Daphniphylli essential oilAfter the liquid is sprayed on the leaf surface for 25 days, the content of the photosynthetic pigment in the bidens bipinnata leaves and the maximum conversion rate of a photosystem II are inhibited to different degrees (figure 6). In general, the content of photosynthetic pigments in the leaves of Bidens bipinnata decreases with the increase of the concentration of the treatment solution, but the decrease degree is different according to the type of the photosynthetic pigments. The chlorophyll-a content was only at the highest concentration treatment (2500 mg/m) compared to the control group²) The content of the protein is obviously reduced, the reduction range is 57.6 percent of that of a control group (figure 6a), and the content of the protein is not obviously different from that of the control group under the treatment of other concentrations. The chlorophyll b content is in the medium-high concentration (1250 mg/m)²，2500mg/m²) The decrease was significant, 24.5% and 57.3% respectively compared to the control group (fig. 6 b). The content of chlorophyll b is reduced under the treatment of medium-low concentration, but has no obvious difference compared with the control group. The carotenoid content in Bidens bipinnata leaves is more sensitive to solutions of essential oil of flea grass than to chlorophyll. At all concentrations, the carotenoid content in the leaves of Bidens pilosa was significantly reduced compared to the control group, and the degree of reduction was graded with the concentration gradient (FIG. 6 c). Wherein, no significant difference (125 mg/m) exists between medium and low concentration treatment groups²，250mg/m²，625mg/m²) No significant difference was observed between the high concentration treatment groups (1250 mg/m)²，2500mg/m²). However, all the treatment groups were significantly reduced compared with the control group, and the reduction was 31.1% (125 mg/m)²)、29.2％(250mg/m²)、30.0％(625mg/m²)、40.3％(1250mg/m²) And 54.5% (2500 mg/m)²)。

Under the foliage spraying treatment of the zoysia essential oil solution, not only the content of the photosynthetic pigment is influenced, but also the conversion efficiency of the light energy in the reaction center of a light system II is obviously inhibited. After 25 days of foliar spray treatment of the essential oil solution of daphnia (figure 6d), the rate of conversion of photosystem II under dark adaptation was determined and it was found that the maximum photochemical efficiency (maximum quantum efficiency of PSII photochemistry, Fv/Fm) of photosystem II decreased with increasing concentration of the treatment solution, and the extent of decrease increased with increasing treatment concentration of the essential oil solution of daphnia (figure 6 d). Of the plant of Bidens pilosa, photosystem II compared with the controlThe maximum photochemical efficiency is respectively reduced by 14.3 percent (125 mg/m)²)、16.8％(250mg/m²)、22.5％(625mg/m²)、30.4％(1250mg/m²) And 43.6% (2500 mg/m)²). 6. Determination of Bidens pilosa antioxidant enzyme activity and malonaldehyde content

And (3) on the 5 th day after the 5 th time of the daphnia grass essential oil leaf surface spraying treatment, taking a plurality of fresh leaves, quickly putting the fresh leaves into liquid nitrogen for freezing storage, and using the fresh leaves for the subsequent determination of the activity of the antioxidant enzyme and the content of malondialdehyde. Wherein, the activity of the Superoxide dismutase (SOD) of the bidens bipinnata leaves is measured by an hydroxylamine method (Oyanagui 1984), the activity of the Catalase (Catalase, CAT) is measured by an ammonium molybdate method (G Louth 1991), the activity of the Peroxidase (POD) is measured by a colorimetric method (Verlander1995), the activity of the Ascorbate Peroxidase (APX) is measured by a colorimetric method (Nakano & Asada 1981), and the content of the Malondialdehyde (Malondialdehydehyde, MDA) is measured by a thiobarbituric acid method (Zhou & Leul 1998). The experiments are all carried out by using a kit (Nanjing institute of bioengineering), and the specific operation steps are completed according to the kit specification.

The experimental results are as follows:

after the treatment of spraying the flea-grass essential oil solution on the leaf surface for 25 days, along with the increase of the treatment concentration, the activity of the antioxidant enzyme of the bidens bipinnata leaves is firstly enhanced and then reduced, and the content of Malondialdehyde (MDA) is increased, and the result is shown in figure 7. Under medium-low concentration treatment (less than or equal to 625 mg/m)²) The activities of SOD, POD and APX were increased, but when the treatment concentration reached 1250mg/m²The activity of this enzyme is significantly inhibited. CAT activity trend was similar to that of the above antioxidant enzymes. At the highest concentration treatment (2500 mg/m)²) The activities of SOD, CAT, POD and APX were all inhibited, and decreased by 11.64%, 36.98%, 21.20% and 28.96% respectively, compared to the control group (FIGS. 7 a-d).

Malondialdehyde is one of the products of cell membrane lipid peroxidation, and the content of malondialdehyde not only represents the degree of membrane lipid peroxidation, but also indirectly reflects the degree of injury of plant cell membranes. From the measurement results of the malondialdehyde content, the foliar spray treatment of the zoysia oil solution can cause serious damage to the bidens bipinnata leaves, and the MDA content is significantly increased compared with the control at all treatment concentrations and is increased along with the increase of the treatment solution concentration (fig. 7 e). Specifically, after the daphnia foenum-graecum essential oil solution is sprayed on the leaf surfaces for 25 days, compared with a control group, the MDA content of the bidens bipinnata leaves is increased by 38.14%, 49.71%, 55.55%, 62.80% and 69.92% under the treatment of 5 concentrations.

In conclusion, through the experiments on the allelopathy of the flea-fleabane essential oil on the growth of the bidens bipinnata, the following results can be obviously seen: the flea-flea essential oil has obvious inhibiting effect on the growth of the bidens bipinnata and can be used as or used for preparing the herbicide of the bidens bipinnata.

While the invention has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. Use of a flea grass extract as or in the preparation of a herbicide to remove Bidens pilosa.

2. The use of a flea grass extract according to claim 1 wherein the flea grass extract is flea grass essential oil.

3. Use of a flea grass extract according to claim 2 wherein the flea grass essential oil comprises: alpha-terpinene, p-cymene, gamma-terpinene, terpinen-4-ol, carvacrol methyl ether, carvacrol, beta-bisabolene and moschus ether A.

4. Use of a flea grass extract according to claim 3,

in the flea-grass essential oil: the content of the alpha-terpinene is 1-7%; the content of p-cymene is 3-18.5%; the content of the gamma-terpinene is 18.0-40.3%; the content of terpinen-4-ol is 0.05-0.6%; the content of carvacrol methyl ether is 3-18%; the content of carvacrol is 12-38%; the content of the beta-bisabolene is 0.7-12%; the moschus ether A content is 0.02-0.1%.

5. The use of the flea grass extract as claimed in claim 3 or claim 4 wherein the flea grass essential oil further comprises α -thujene, α -pinene, β -pinene, myrcene, α -phellandrene, 3-carene, limonene, cis-ocimene, isoterpinolene, fenchytone, linalool, borneol, α -terpineol, thymol, trans-caryophyllene, farnesene, lupinene, β -sesquiphellandrene, and caryophyllene oxide II.

6. Use of a flea grass extract according to claim 5,

in the flea-grass essential oil: the content of the alpha-arborvitae is 1.4-2.3%; the content of the alpha-pinene is 0.1-0.45%; the content of the beta-pinene is 0.13-0.15%; the content of myrcene is 0.08-0.18%; the content of the alpha-phellandrene is 0.07-0.155 percent; the content of the 3-carene is 1.5-2.4%; the content of the limonene is 1.9-3.5%; the content of the cis-ocimene is 0.08-0.16%; the content of the terpinolene is 0.2-2.3%; the content of the fenchone is 0.1-0.42%; the linalool content is 0.02-0.095%; the content of the borneol is 0.07-0.45%; the content of the alpha-terpineol is 0.05-0.19%; the content of thymol is 0.45-0.75%; the content of the trans-caryophyllene is 0.35-0.67%; the content of the farnesene is 0.04-0.099%; the content of the lupinene is 0.4-1.993%; the content of the beta-sesquioenanthe is 0.15-0.291%, and the content of the caryophyllene oxide II is 0.120-0.29%.

7. Use of a flea grass extract according to any one of claims 2 to 6 wherein the process for the preparation of the essential oil of flea grass comprises the steps of: extracting oil from the flea grass by adopting essential oil distillation equipment to obtain the flea grass essential oil.

8. A herbicide, wherein said herbicide comprises a flea grass extract.

9. A herbicide formulation as claimed in claim 8, wherein the flea grass extract is flea grass essential oil.

10. The herbicide as claimed in claim 9, wherein the concentration of essential oil of flea grass in the herbicide is 5 μ L/L to 30 ml/L.