CN114287422A - Application of limonene in preparation of tobacco seed germination promoter or inhibitor - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of limonene in preparation of a tobacco seed germination promoter or inhibitor. The invention researches the action of active substances in marigold roots and stems extracted by various extraction methods on ralstonia solanacearum, determines that limonene and alpha-terthiophene in the marigold roots and stems are active substances with the effect of inhibiting bacterial wilt, and provides a corresponding preparation method. Further verifying the control effect of limonene and alpha-terthiophene on ralstonia solanacearum HF-1-1 in a tobacco pot experiment, the inhibition effect of the alpha-terthiophene on HF-1-1 is 28.63% in the 9 th week of tobacco seedling growth, and the control effect of limonene on HF-1-1 reaches 62.27%. Thus laying the foundation for preventing and controlling the soil-borne diseases of the tobacco by the botanical fungicide substance, namely the marigold rhizome extract limonene.

Description

Application of limonene in preparation of tobacco seed germination promoter or inhibitor

The application is a divisional application of an invention patent with application number 202010774068.0, and the application date of the original application is as follows: 2020-08-04; the invention creates the name: active ingredients of marigold rhizome and application thereof in preventing and treating ralstonia solanacearum.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of limonene in preparation of a tobacco seed germination promoter or inhibitor.

Background

Tobacco is one of the main economic crops in China, and because tobacco fields are continuously planted for many years and chemical fertilizers and pesticides are applied in large quantities, tobacco diseases and insect pests, particularly soil-borne diseases are serious, and the healthy development of the tobacco industry is severely restricted. The traditional chemical agent has poor prevention effect on soil-borne diseases of tobacco, is easy to generate drug resistance, and easily causes environmental pollution problems such as drug residue and the like.

Black shank (Tobacco black shank) and bacterial wilt (Tobacco bacterial wilt) are common diseases of Tobacco plants in mass and flourishing for a long time, and are expressed by withering of Tobacco plants, yellowing of leaves and blackening of stems. Tobacco bacterial wilt often occurs together with tobacco black shank and root knot nematode, and can cause the tobacco in the whole field to die when serious, thereby causing huge economic loss to tobacco growers. The biological control fungicide for the tobacco soil-borne diseases is developed, popularized and applied, the application of chemical agents is avoided or reduced, and the biological control fungicide has practical production significance for reducing the occurrence of the tobacco soil-borne diseases, guaranteeing the planting yield and output value of tobacco growers and the planting enthusiasm of the tobacco growers and reducing environmental pollution. The botanical pesticide has the advantages of no residue, low toxicity, difficult generation of drug resistance, easy mixing with other medicaments and the like, and has become one of the hot spots of domestic and foreign research in recent years.

Many researches find that marigold has better inhibiting or poisoning effect on various pathogenic microorganisms. The marigold has wide application prospect in the aspects of preventing and controlling tobacco soil-borne diseases, particularly bacterial wilt, black shank and the like, and is particularly important for deeply researching the disease resistance mechanism of the marigold. The existing researches mainly focus on the influence of crude extracts of different parts on the hypha growth and spore germination of pathogenic bacteria and the measurement of the difference of some physiological and biochemical indexes of different growth periods of infected plants treated by the crude extracts and control samples. The researches on the distribution, specific physicochemical properties, the relationship between the structure and the activity, the bactericidal and bacteriostatic action mechanisms and the like of active ingredients in the plant body are basically blank. Besides, marigold is used as a plant resource for large-area planting, the collected part is mostly a flower part, and the non-flower part which accounts for the major part of the whole plant is not utilized well all the time.

Previous researches find that marigold flowers have good biological activity on tobacco root-knot nematode disease. In actual life, marigold flowers are generally used for extracting lutein and have been well applied. The leaf, stem and root are generally burnt together with straws and the like, which not only pollutes the environment, but also causes great waste of resources, so that the problems that the bactericidal activity of the leaf, stem and root of marigold is fully researched and utilized in the biological control of tobacco bacterial wilt in tobacco fields, the components of marigold extract and substances in the components have the antibacterial effect, the structural composition is what, how to utilize the extract to the maximum degree and the like are urgently needed to be solved.

Disclosure of Invention

In view of the problems in the prior art, the invention provides an application of limonene in preparation of a tobacco seed germination promoter or inhibitor, and aims to solve part of the problems in the prior art or at least alleviate part of the problems in the prior art.

The preparation method of the ralstonia solanacearum antibacterial liquid comprises the steps of cleaning and drying roots and stems of marigold, grinding the roots and stems into powder, and extracting the powder by using petroleum ether or ethyl acetate, wherein the mass-volume ratio of the powder to the petroleum ether or the ethyl acetate is 1:20, and the extraction time is 5 hours.

The invention also provides a preparation method of the ralstonia solanacearum bacteriostat, which comprises the steps of passing the marigold rhizome aqueous extract through a 0.22 mu m filter membrane, carrying out HPLC separation, and preparing acetonitrile, 0.1 percent trifluoroacetic acid (TFA) and water, 0.1 percent trifluoroacetic acid (TFA) as mobile phases; a short column of chromatography column C18; the flow rate is 0.7 ml/min; the column temperature is 25 ℃; ultraviolet detection wavelength is 330 nm; the elution procedure was:

collecting substances for 39-46min to obtain bacterial inhibitor of Ralstonia solanacearum.

Further, the ralstonia solanacearum bacteriostat comprises at least one of limonene and alpha-terthiophene.

Further, the preparation method of the marigold rhizome water extract comprises the steps of putting the marigold rhizome powder into distilled water, oscillating for 2 hours in a shaking table at 30 ℃ and 230rpm, then oscillating for 1 hour in ultrasonic waves, then putting into a 50 ℃ constant-temperature water bath kettle for water bath for 2 hours, and finally standing overnight; filtering the soaking solution with filter membrane, and placing the filtered solution into a low-temperature concentration instrument to obtain marigold rhizome water extract.

The invention also provides application of the limonene in preparation of a reagent for preventing and treating ralstonia solanacearum.

The invention also provides application of the alpha-terthiophene in preparation of a reagent for preventing and treating ralstonia solanacearum.

The invention also provides application of the limonene in preparation of a tobacco seed germination promoter or inhibitor.

The invention also provides application of the alpha-terthiophene in preparation of a tobacco seed germination inhibitor.

In summary, the advantages and positive effects of the invention are:

the plant is a natural treasury of bioactive compounds, and the root, stem, leaf and flower of the plant contain various antibacterial and bactericidal active ingredients. At present, there are new biological agents which are prepared by extracting effective components for resisting fungi, bacteria, viruses and nematodes from plants and processing the extracted monomer substances as lead compounds. The botanical antibacterial agent is natural, has the characteristics of easy degradation, low residue, environmental compatibility and the like, and meets the requirements of economic ecological sustainable development. Therefore, by taking plants as action targets, a new active lead compound is searched from the action targets to become an important way for preventing and treating the microbial diseases of the economic crops, and the method has good development opportunity and huge market space.

Marigold contains various antibacterial and bactericidal active substances. The light activated insecticidal substance alpha-terthiophene and the bacteriostatic substance for the fusarium oxysporum in the root extract have been reported. The research is to extract active substances of marigold roots and stems to inhibit the soil-borne diseases of bacterial wilt and black shank of tobacco, and aims to improve the yield of the tobacco and better develop a plant antibacterial agent. Has profound significance.

The invention researches the action of active substances in marigold roots and stems extracted by various extraction methods on ralstonia solanacearum, determines that limonene and alpha-terthiophene in the marigold roots and stems are active substances with the effect of inhibiting bacterial wilt, and provides a corresponding preparation method. Thus laying the foundation for preventing and controlling the soil-borne diseases of the tobacco by the botanical fungicide substance marigold rhizome extract.

Drawings

FIG. 1 shows the inhibition of HF-1-1 by organic solvent extraction of marigold roots;

FIG. 2 is a graph of ion peaks of GC-MS of marigold root, stem and leaf;

FIG. 3 is a plate bacteriostasis experiment of ethanol and water extracts of marigold rhizome on HF-1-1;

FIG. 4 shows the result of TLC plate separation of an aqueous extract of marigold rhizome;

FIG. 5 is a TLC run-plate validation after column chromatography separation;

FIG. 6 is a graph of ion peaks from HPLC separation of marigold rhizome aqueous extract;

FIG. 7 shows the inhibition of HF-1-1 by HPLC separation of marigold aqueous extract;

FIG. 8 is a graph of ion peaks from HPLC separation of marigold rhizome aqueous extract;

FIG. 9 is an infrared spectrum of substance 1;

FIG. 10 is an infrared matching plot of substance 1;

FIG. 11 is a nuclear magnetic hydrogen spectrum of substance 1;

FIG. 12 is a nuclear magnetic carbon spectrum of substance 1;

FIG. 13 is a gas mass spectrometry total ion flow diagram of Mass 1;

FIG. 14 is a library match of the peak at 6.737 min;

FIG. 15 is the result of elemental analysis of substance 1;

FIG. 16 is a scanning electron micrograph;

FIG. 17 is an infrared spectrum of substance 2;

FIG. 18 is an infrared matching plot for substance 2;

FIG. 19 is a nuclear magnetic hydrogen spectrum of substance 2;

FIG. 20 is a nuclear magnetic carbon spectrum of substance 2;

FIG. 21 is a gas mass spectrometry total ion flow diagram of Mass 2;

FIG. 22 is a library match plot of the peak at 18.201 min;

fig. 23 is the elemental analysis result of substance 2:

FIG. 24 is a scanning electron micrograph;

FIG. 25 is a plate bacteriostasis experiment of limonene on HF-1-1;

FIG. 26 is a plate bacteriostasis experiment of alpha-terthiophene and limonene on HF-1-1;

FIG. 27 is a graph demonstrating the bacteriostatic effects of limonene and alpha-terthiophene on HF-1-1;

FIG. 28 is a graph of the inhibition of HF-1-1 by various amounts of limonene;

FIG. 29 is the effect of limonene on HF-1-1 growth;

FIG. 30 is the effect of α -terthiophene on HF-1-1 growth;

FIG. 31 is the statistics of the incidence and disease index of tobacco bacterial wilt after different active substance treatments; DI is the incidence and DI is the disease index.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The invention discloses an application of limonene in preparation of a tobacco seed germination promoter or inhibitor. The marigold used in the invention is marigold grown in a greenhouse for 4-6 months. The details of the present invention are shown in the following examples.

Example 1 marigold rhizome active substance assay

1. Organic solvent analysis of marigold rhizome active substance

Cleaning marigold rhizome, oven drying, and grinding into powder. 4 solvents of petroleum ether, chloroform, ethyl acetate and n-butanol are selected to be respectively subjected to Soxhlet extraction. 4g of rhizome powder and 80ml of solvent are extracted by a Soxhlet extractor for 5 hours respectively. Then concentrating the extract, adding 1% of tobacco bacterial wilt pathogen HF-1-1 fermentation broth into the NA solid culture medium with TTC, punching a hole with the diameter of 5mm on a flat plate, adding 100 μ L of solvent extraction agent, placing the flat plate in an incubator at 37 ℃ for 12h, and taking each extract as a blank control. And observing the growth condition of the flat plate.

The results are shown in FIG. 1, where a is ethyl acetate extract, b is petroleum ether extract, c is chloroform extract, d is petroleum ether extract, f is chloroform extract, and g is ethyl acetate extract. It can be known that the petroleum ether and the petroleum ether extract have no inhibiting effect on HF-1-1; chloroform and chloroform extract have certain inhibiting effect on HF-1-1, and substances in the chloroform or chloroform extract are presumed to have effect on HF-1-1 by combining the first experiment; the ethyl acetate and the extract thereof have no obvious inhibition effect on HF-1-1. By combining the analysis of the inhibition effect of different solvent extraction liquids on the ralstonia solanacearum, the highest inhibition rate of the petroleum ether extraction liquid on the ralstonia solanacearum reaches 89.16%, and the highest inhibition rate of the ethyl acetate extraction liquid on the ralstonia solanacearum reaches 89.59%.

2. GC-MS method for analyzing active substances of different parts of marigold

Removing the root of marigold, cleaning, soaking the root, stem and leaf in sterile water for 48 hr, and extracting with ethyl acetate; vacuum concentrating the extractive solution into extract, adding 80 μ L of 20mg/ml methoxylamine pyridine hydrochloride, ultrasonically oscillating for 5min, oximating in 37 deg.C incubator for 120min, adding 10 μ L of internal standard solution and 100 μ L of MSTFA solution, derivatizing in 60 deg.C oven for 3h, filtering with injector into liner tube, placing the liner tube into gas bottle, and loading to obtain gas. GC conditions were as follows: helium gas with flow rate of 1ml/min, inlet temperature of 235 deg.C, ion source temperature of 250 deg.C, interface temperature of 260 deg.C, temperature raising program of 50 deg.C for 2min, raising to 180 deg.C at speed of 5 deg.C/min, raising to 280 deg.C at speed of 15 deg.C/min, and eliminating solvent peak at solvent cutting time of 4.5 min. Each treatment was in triplicate.

The results are shown in FIG. 2, in which a, b and c are GC-MS ion peak diagrams of marigold roots, stems and leaves, respectively. The ion peak distribution of the root, stem and leaf of marigold is greatly different. GC-MS analysis results show that the marigold root, stem, leaf and root secretion contain a plurality of plant natural components, wherein the marigold root mainly comprises: amines, esters, aldehydes, alkanes, ketones, acids, alkenes, alcohols, imidazoles, triazines, thiophenes and furans, in proportions of 3.144%, 56.152%, 0.136%, 0.503%, 1.054%, 0.066%, 0.003%, 8.33%, 0.005%, 0.033%, 30.21% and 0.364%, respectively; the marigold stems mainly comprise: amines, esters, aldehydes, alkanes, ketones, acids, alkenes, alcohols, imidazoles, triazines, thiophenes and furans in the proportion of 1.766%, 43.339%, 0.314%, 11.908%, 1.166%, 0.19%, 0.012%, 11.869%, 0.267%, 0.055%, 28.349% and 0.765% respectively; the marigold leaf mainly comprises: amines, esters, aldehydes, alkanes, ketones, acids, alkenes, alcohols, imidazoles, triazines, thiophenes and furans, which account for 3.403%, 42.948%, 0.789%, 15.321%, 0.932%, 0.837%, 0.135%, 18.045%, 0.763%, 0.064%, 3.045% and 13.72% respectively; the marigold root secretion mainly comprises: amines, esters, aldehydes, alkanes, ketones, acids, alkenes, alcohols, thiophenes, furans and saccharides account for 5.149%, 14.503%, 0.502%, 9.858%, 0.307%, 56.054%, 0.127%, 8.349%, 2.668%, 0.198% and 2.286%, respectively. It is known that the difference in substance content in each part of marigold is large.

3. Analysis of bacteriostatic effect of marigold rhizome water-soluble extract

The marigold is pulled up and cleaned, crushed by a grinding machine and sieved by a 60-mesh sieve. Weighing 5g of marigold rhizome powder, putting into 100mL of distilled water, shaking for 2h at 30 ℃ and 230rpm, then putting into ultrasonic waves, shaking for 1h, then putting into a 50 ℃ constant temperature water bath kettle, carrying out water bath for 2h, and finally standing overnight. Filtering the soaking solution with a filter membrane, and placing the filtered solution into a low-temperature concentration instrument to obtain 20mL of concentrated solution which is the marigold rhizome aqueous extract.

As shown in a plate experiment of inhibiting ralstonia solanacearum HF-1-1 by using an ethanol extract and a water extract of marigold rhizomes, the ethanol extract of the marigold rhizomes has no inhibition effect on HF-1-1, while the water extract of the marigold rhizomes has obvious inhibition effect on HF-1-1, and the inhibition ring phi of the ethanol extract of the marigold rhizomes is 15.324 +/-0.311 mm (as shown in a figure 3, wherein a1 and a2 are control ethanol, a3 and a4 are plate inhibition experiments of the ethanol extract of the marigold rhizomes on HF-1-1, b1 and b2 are control water, and b3 and b4 are plate inhibition experiments of the water extract of the marigold rhizomes on HF-1-1). It is presumed that the active bacteriostatic component of marigold rhizome extract is very soluble in water.

4. TLC thin layer chromatography analysis of marigold rhizome aqueous extract

Cutting the silica gel chromatography plate into a rectangular plate with the length of 5cm and the width of 2 cm; sucking a small amount of marigold rhizome water extract to the bottom end of the silica gel plate by using a capillary sample application tube with the diameter of 1mm, and repeating the process for three times; preparing different spreading agents, pouring the spreading agents into a spreading cylinder, putting the well-spotted silica gel plate into the spreading agents, taking out the spreading agents when the spreading agents reach the top end, drying the spreading agents by blowing, and placing the spreading agents under an ultraviolet lamp with the wavelength of 365nm to observe strips on the silica gel plate.

Water extract of marigold rhizome; a glass capillary tube having an inner diameter of 0.5 mm; a silica gel plate; acetone; absolute ethyl alcohol; hydrochloric acid; n-butanol; chloroform; dichloromethane; acetic acid; formic acid; ethyl acetate; methanol; n-hexane. Through consulting the literature, the method for finding out the TLC point plate spreading agent with the high polarity is adjusted to obtain the optimal ratio of the spreading agent. The bands of the silica gel plate were observed under 365nm UV. As shown in fig. 4, wherein (a) is acetone: anhydrous ethanol: hydrochloric acid 5: 3: 0.5; (B) n-butanol: acetic acid: ethanol: water is 4:1:1: 2; (C) chloroform: methanol: water: formic acid: glacial acetic acid 15: 5: 0.5: 1: 1; (D) dichloromethane: acetic acid: ethanol: water 5: 1:1: 0.5; (E) acetone: methanol: acetic acid 5: 3: 1; (F) n-butanol: acetic acid: ethanol: water 5: 1: 2: 1; (G) dichloromethane: methanol 3: 1; (H) dichloromethane: methanol: formic acid 2: 1: 1; (I) n-butanol: acetic acid: ethanol: water-4: 1:1: 1; (J) dichloromethane: acetic acid: ethanol: water-4: 1: 1; (K) n-butanol: acetic acid: ethanol: water 5: 1:1: 1.5; (L) Ethyl acetate: acetic acid: ethanol: water-4: 1:1: 1.5; (M) chloroform: methanol: water 5: 3: 1; (N) N-butanol: acetic acid: ethanol: water 5: 1:1: 1.2; (O) n-butanol: acetic acid: ethanol: water 6: 1:1: 1.5; (P) n-butanol: acetic acid: ethanol: water-8: 1:1: 2; (Q) methanol: water-8: 2; (R) n-hexane: ethyl acetate: acetic acid 5: 3: 1; (Y) n-hexane: ethyl acetate: acetic acid 7: 2: 1. the separation effect of the method R is best, and 5 types of compounds are separated. The formula of the optimal developing agent is finally obtained by n-hexane: ethyl acetate: acetic acid 5: 3: 1.

5. silica gel column chromatography analysis of marigold rhizome aqueous extract

Drying the sample and weighing; dissolving the sample with an organic solvent (3-5 mL); mixing silica gel powder with the same weight as the sample on a water bath kettle (the water temperature is not too high), volatilizing the solvent, and fully and uniformly adsorbing the sample on the silica gel powder; adding silica gel powder 10 times the amount of the sample into the silica gel column, and pumping with a vacuum pump for 20min to make the silica gel powder compact, and plugging a small cotton ball at the lowest end of the silica gel powder to prevent the silica gel powder from overflowing; adding the uniformly stirred sample powder into a silica gel column, uniformly spreading the sample powder, adding silica gel powder with the thickness of 1-2cm, and finally filling a cotton ball; adding n-hexane solvent with minimum polarity into a column by volume to wet silica gel powder in the column and fill column filler; gradient elution procedure: n-hexane: ethyl acetate: acetic acid 50: 3: 1 (Single column volume)

N-hexane: ethyl acetate: acetic acid 30: 3: 1(2 column volume)

N-hexane: ethyl acetate: acetic acid 20: 3: 1(2 column volume)

N-hexane: ethyl acetate: acetic acid 10: 3: 1(2 column volume)

N-hexane: ethyl acetate: acetic acid 5: 3: 1(3 column volume)

N-hexane: ethyl acetate: acetic acid 1:1: 1(2 column volume)

The layered samples were connected with a test tube.

As shown in FIG. 5, wherein a is test tube sample strips numbered 1-14; b is a test tube sample strip of 15-22; c is a test tube sample strip for verification 23-24; d is a test tube sample strip of 25-30. After column chromatography, TLC plates verified that distinct bands were clearly separated, indicating that different classes of material were separated.

6. HPLC separation method for marigold rhizome aqueous extract

2mL of marigold rhizome aqueous extract is filtered through a 0.22 mu m filter membrane; acetonitrile + 0.1% trifluoroacetic acid (TFA) and water + 0.1% trifluoroacetic acid (TFA) were prepared as mobile phases; a short column of chromatography column C18; the flow rate is 0.7 ml/min; the column temperature is 25 ℃; the ultraviolet detection wavelength is 330 nm. The elution procedure was as follows:

as shown in FIG. 6, after HPLC separation, various components were separated from the aqueous extract of marigold rhizome, and the separated substances were collected in stages: 32-37.01min, 39-46min, 51.04-56.05min and 58.03-62.05 min. And finally, carrying out a plate bacteriostasis experiment on the collected substance by using ralstonia solanacearum HF-1-1.

FIG. 7, wherein a is 32-37.01 min; b is 39-46 min; c is 51.04-56.05 min; d is 58.03-62.05 min. The diameter of the substance inhibition ring is 15.1 +/-0.057 mm at 32-37.01 min; the diameter of the substance inhibition ring is 21.3 +/-0.22 mm within 39-46 min; 51.04-56.05min of substance inhibiting ring with diameter of 10.4 + -0.08 mm; 58.03-62.05min of substance inhibiting ring diameter of 12.32 + -0.062 mm. It is known that the substance at 39-46min has the best effect of inhibiting HF-1-1 (T-test, p < 0.05).

7. GC-MS method for analyzing 39-46min effective antibacterial component of marigold rhizome water extract

Spin-drying the antibacterial substance for 39-46min, adding 80 μ L of 20mg/ml methoxylamine pyridine, performing ultrasonic oscillation for 5min, placing in a 37 ℃ incubator for oximation for 120min, adding 10 μ L of internal standard solution and 100 μ L of MSTFA solution, placing in a 60 ℃ oven for derivatization for 3h, filtering with a syringe, placing in a gas-phase bottle, and detecting. GC conditions were as follows: helium gas with flow rate of 1ml/min, inlet temperature of 235 deg.C, ion source temperature of 250 deg.C, interface temperature of 260 deg.C, temperature raising program of 50 deg.C for 2min, raising to 180 deg.C at speed of 5 deg.C/min, raising to 280 deg.C at speed of 15 deg.C/min, and eliminating solvent peak at solvent cutting time of 4.5 min.

The substance powder detected by GC-MS is amines, alkanes, acids, alkenes, triazines, ketones, esters, alcohols and furans. Wherein the ester substances account for 77.438% of the total substances at most; the proportion of the amines, alkanes, acids, alkenes, triazines, ketones, alcohols and furans is 0.525%, 0.35%, 5.502%, 0.564%, 0.019%, 0.689%, 14.837 and 0.075% in sequence.

Further separating 39-46min substance of marigold rhizome water extract by HPLC, detecting the part with antibacterial effect by GC-MS, and detecting the substances as shown in the following table. 2 amine substances, 2 alkane substances, 4 acid substances, 1 alkene substance, 1 triazine substance, 2 thiophene substances, 3 ketone substances, 8 ester substances, 2 alcohol substances and 2 furan substances are obtained.

TABLE 1 GC-MS method for analyzing effective antibacterial ingredient classification of marigold rhizome aqueous extract

Separating the part with bacteriostatic effect by HPLC for several times, collecting a large amount of the separated part until obtaining a relatively pure peak, performing plate bacteriostatic verification on the substances with peak time of 1.852min and 2.654min, and finding that the substances have good bacteriostatic effect.

8. Structural analysis of active ingredients

The substances with peaks at 1.852min (substance 1) and 2.654min (substance 2) in FIG. 8 and the processed ralstonia solanacearum are sent to Shanghai Fuda detection technology group Limited for analysis such as nuclear magnetism, infrared ray, mass spectrum, elemental analysis, and scanning electron microscope.

Substance 1:

as shown in fig. 9, the infrared spectrum of substance 1 was obtained, and the detection results were matched with the database to obtain a matching result of limonene of 99.44% (fig. 10).

And then detecting a nuclear magnetic hydrogen spectrum (figure 11), a nuclear magnetic carbon spectrum (figure 12), a gas mass spectrum total ion flow diagram (figure 13) and a spectrum library matching diagram of a peak at 6.737min (figure 14) of the substance 1, and further verifying that the substance 1 is the limonene. The elemental analysis results for substance 1, as shown in fig. 15, match the elemental composition of limonene, again verifying that substance 1 is limonene.

FIG. 16a is a scanning electron micrograph of an untreated ralstonia solanacearum bacterial solution showing that the cells are uniformly distributed and have an oblong shape; in the scanning electron micrograph of the bacterial liquid of Ralstonia solanacearum treated with substance 1, the cells are disordered and unevenly distributed, and are mostly small oval (FIG. 16 b). As can be seen from the above, substance 1 has a large influence on the growth of bacterial wilt bacteria.

Substance 2:

as shown in fig. 17, the infrared spectrum of substance 2 was obtained, and the detection results were matched with the database to obtain a matching result of α -terthiophene of 99.44% (fig. 18).

And then detecting a nuclear magnetic hydrogen spectrum (figure 19), a nuclear magnetic carbon spectrum (figure 20), a gas phase mass spectrum total ion flow diagram (figure 21) and a spectrum library matching diagram (figure 22) of a peak at 18.201min of the substance 2, and further verifying that the substance 2 is alpha-terthiophene.

The elemental analysis results for substance 2, shown in fig. 23, match the elemental composition of α -terthiophene, again verifying that substance 2 is α -terthiophene.

FIG. 24a is a scanning electron micrograph of an untreated ralstonia solanacearum bacterial solution showing that the cells are uniformly distributed and have an oblong shape; in the scanning electron micrograph of the bacterial liquid of Ralstonia solanacearum treated with substance 2, the cells are disordered and unevenly distributed, and are mostly small oval (FIG. 24 b). As a result, substance 2 had a large influence on the cell formation of bacterial wilt bacteria.

By combining the analysis of the substance 1 and the substance 2 such as nuclear magnetism, infrared ray, mass spectrum, element analysis, scanning electron microscope and the like and the detection result of GC-MS, the limonene and the alpha-terthiophene in the marigold rhizome extract have bactericidal activity, so that the two compounds are selected to further verify the bacteriostatic action of the marigold rhizome extract on tobacco bacterial wilt and black shank.

Example 2 analysis of bacteriostatic Effect of marigold rhizome active ingredient

Reagent medicine and bacterial related to this example: tobacco bacterial wilt pathogen (Ralstonia solanacearum), Phytophthora parasitica (Phytophthora nicotianae parasitica var) (laboratory deposited strain); limonene and alpha-terthiophene were purchased from the Shanghai Michelle chemical technology, Inc. Tobacco variety: the tobacco variety tested was Yunyan 86.

1. Determination of bacteriostatic activity of active ingredients of marigold rhizome

The sterilized 150mL nutrient agar solid culture medium is heated and melted completely. Cooling to about 55 ℃, pouring into culture dishes with 15mL per dish, and solidifying for later use. A punch method is adopted to punch holes with the diameter of 5mm on NA culture medium with the diameter of 90mm, and 4 holes are punched on each dish. mu.L of limonene was added drop wise to the wells, sterile water was used as a control. After the media had absorbed the drug sufficiently, the plates were transferred to a fume hood to a concentration of 1.0x10⁸The CFU/mL bacterial suspension of the ralstonia solanacearum is uniformly sprayed on the surface of the medicament flat plate, so that the atomized bacterial suspension is naturally scattered on the medicament flat plate. And (3) sealing after the flat plate fully absorbs the bacterial suspension, inverting the flat plate and culturing in a constant-temperature incubator at 37 ℃, checking the bacteriostatic effect after 24 hours, and measuring the diameter of the bacteriostatic zone by using a cross method. The size of the zone of inhibition was recorded and repeated 3 times.

According to GC-MS and literature research results, primarily speculating antibacterial effective components of limonene and alpha-terthiophene in marigold rhizome water extract, adding 1% HF-1-1 fermentation liquor into an NA solid culture medium mixed with TTC, uniformly mixing, punching a hole with the diameter of 5mm, adding limonene and alpha-terthiophene into the hole, putting a flat plate into an incubator at 37 ℃ for constant temperature culture for 24h, and observing the size of an antibacterial ring. Limonene has an inhibitory effect on ralstonia solanacearum (HF-1-1) (fig. 25, wherein a1 and a2 are 20 μ L limonene, a3 is 50 μ L limonene, b1 and b3 are 50 μ L α -terthiophene, b2 is 20 μ L α -terthiophene), limonene at different concentrations has a significant difference in inhibitory effect on ralstonia solanacearum (HF-1-1), 50 μ L limonene stock solution has a strong inhibitory effect on HF-1-1, and the inhibition ring Φ is 19.31 ± 0.214 mm; 20 μ L of limonene gave an HF-1-1 inhibition zone Φ of 13.31 ± 0.159 mm.

As shown in FIG. 26, a is 50. mu.L of α -terthiophene, and b is a 40. mu.L of limonene inhibits HF-1-1 in a plate experiment. The colony numbers of the ralstonia solanacearum of a and b are respectively 2.15x10⁷CFU/mL and 1.08x10⁷CFU/mL. It is found that the effect of limonene on inhibiting HF-1-1 is stronger than the effect of alpha-terthiophene on inhibiting HF-1-1.

Considering that the dilution of the plate with HF-1-1 may not be uniform, it was tried to grow HF-1-1 by pouring it directly into the medium, and then punch the hole and add the extract, and observe the zone of inhibition. The inhibitory effect of limonene on HF-1-1 was again examined in the same manner. FIG. 27, wherein a1 is 20 μ L limonene, a2 is 10 μ L limonene, a3 is control ethanol; b1 was 10 μ L α -terthiophene, b2 was 20 μ L α -terthiophene, b3 was control ethanol. Ethanol has no inhibition effect on the growth of HF-1-1, and both limonene and alpha-terthiophene have inhibition effect on HF-1-1.

In order to further confirm the inhibition effect of limonene on bacterial wilt pathogenic bacteria, a flat plate experiment and a shaking flask fermentation experiment are carried out again. mu.L, 20. mu.L, 30. mu.L and 40. mu.L of limonene were added to the plate mixed with Ralstonia solanacearum HF-1-1, respectively. After being cultured in a 37 ℃ constant temperature incubator for 12 hours, the colony numbers of the ralstonia solanacearum are 543.67 +/-64.45, 378.67 +/-48.95, 164.67 +/-53.51 and 108 +/-38.35 (10)⁵CFU/mL) (FIGS. 28a, b, c, d), it is known that the bacteriostatic effect on HF-1-1 is increased with increasing amount of limonene. And the colony number of the ralstonia solanacearum has obvious difference among different marigold extract dosage (T-test, p)<0.05, fig. 28).

As can be seen from FIG. 29, limonene has a strong inhibitory effect on HF-1-1, and the inhibitory effect increases with increasing dosage. Notably, when the amount of limonene reached 50 μ L, HF-1-1 hardly grew, indicating that 50 μ L of limonene was sufficient to inhibit the growth of HF-1-1. The effect of limonene in marigold rhizome extract on inhibiting bacterial wilt pathogenic bacteria is found to be obvious for the first time.

As can be seen from FIG. 30, α -terthiophene has a certain inhibitory effect on HF-1-1, and α -terthiophene of 100mg/L and 400mg/L has no strong inhibitory effect on HF-1-1 than α -terthiophene of 800 mg/L; the effect of inhibiting HF-1-1 by 800mg/L alpha-terthiophene is obviously lower than that of limonene.

2. Toxicological test of active ingredient compounds of marigold rhizome

The active substance component is subjected to seed germination tests according to 5 concentration gradients of 100%, 75%, 50%, 25% and 0%, each treatment is set to be 3 times, the promotion rate or the inhibition rate of each concentration on the germination of the crop seeds is calculated by using a formula by taking the bud length as an index and taking the 0% concentration as a control.

The promotion rate (inhibition rate)%, i.e. (average value of germination number of treated group-average value of germination number of control group)/average value of germination number of control group × 100%

TABLE 2 Effect of alpha-Trithiofuran on tobacco seed Germination

Under the condition of the concentration of 50 percent or below, the alpha-terthiophene has no obvious inhibition or promotion effect on the germination of the tobacco seeds, but the alpha-terthiophene with high concentration has obvious inhibition effect on the germination of the tobacco seeds, and the negative sign indicates the inhibition.

TABLE 3 influence of limonene on germination of tobacco seeds

Under the condition of the concentration of 50% or below, limonene promotes the germination of tobacco seeds, and limonene with the concentration of 75% or above has an obvious inhibiting effect on the germination of the tobacco seeds; negative signs indicate inhibition.

3. Indoor potting verification of active ingredients of marigold rhizome

When the tobacco seedlings are potted, ralstonia solanacearum pathogenic bacteria are respectively added as a Control (CK), limonene (50 mu L/L) and the addition amount of alpha-terthiophene is 500 mg/L. And (5) beginning to count the incidence rate and disease index of bacterial wilt after 1, 3, 5, 7 and 9 weeks of transplantation. The indoor pot experiment was verified 5 times each time with 3 replicates each of 10 replicate tobacco plants.

The tobacco varieties to be tested are flue-cured tobacco Yunyan 86 and K236, the tobacco is sown and planted in a flowerpot and is placed in an insect cage to prevent pests from eating and injuring, no pesticide is used in management, and only manual watering is carried out when needed. Sowing tobacco of different varieties into seedling pots, covering with film for moisturizing, transplanting into a sterile nutrition pot after seedling emergence, culturing in a 30 ℃ biochemical incubator, and selecting growth vigor when 3-4 true leaves grow out from the tobaccoConsistent tobacco is ready for use. Injecting with a syringe needle into vascular bundle at tobacco root-stem junction, slightly lifting the needle, and inoculating with 0.1mL of vaccine with concentration of 1.0x10¹⁰CFU/mL bacterial suspension of Ralstonia solanacearum, and sterile water is used as a control.

In another experiment, 50mL of single medicament and compound components with different dilution times are irrigated to each plant except for root injury inoculation of pathogenic bacteria. Each 10 tobacco seedlings were treated and repeated 3 times. According to the industry standard 'tobacco disease grading and investigating method' (YC/T39-1996), investigating and recording the disease grade and the disease condition of tobacco plants, and calculating the morbidity and the disease condition index.

See danhenping et al (danhenping, 2003# 16). Selecting a tobacco field with serious tobacco bacterial wilt occurrence and harm all the year round. The effective component extracts with different concentrations are respectively applied after the tobacco seedlings are transplanted. Disease condition investigation is carried out in the disease outbreak period, and the disease incidence and the prevention and treatment effect are respectively calculated according to a formula 1 and a formula 2.

Incidence (%): number of diseased plants/total number of plants × 100 (1)

Control effect (%) - (control area incidence-treatment area incidence)/control area incidence × 100 (2)

After the tobacco is inoculated with the bacterial wilt pathogenic bacteria for 1, 3, 5, 7 and 9 weeks, the incidence rate and disease index statistics of the bacterial wilt show that: after the limonene and the alpha-terthiophene are treated, the incidence rate and the disease index of the bacterial wilt are obviously reduced (figure 31), and particularly, the limonene has the best effect of preventing and treating the bacterial wilt (table 4).

TABLE 4 control of bacterial wilt by marigold active substance (%)

	1	3	5	7	9
						Alpha-terthiophene (di)	0.00	9.56	33.02	44.00	28.63
Limonene (di)	0.00	31.29	49.60	64.35	62.27

Through the analysis, limonene and alpha-terthiophene in marigold rhizomes are basically determined to be active substances capable of inhibiting bacterial wilt.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (1)

1. Application of limonene in preparing a germination promoter or inhibitor for tobacco seeds.

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