Melaleuca alternifolia extract, preparation method and application of melaleuca alternifolia extract in prevention and treatment of tobacco brown spot

Abstract

The invention discloses a melaleuca alternifolia extract, a preparation method and application of the melaleuca alternifolia extract to prevention and treatment of tobacco brown spot, and relates to the technical field of agricultural prevention and treatment, a compound is extracted from melaleuca alternifolia by adopting a steam distillation method, and the chemical component of the compound is identified as terpinen-4-o through gas chromatography-mass spectrometry (GC-MS) combined analysis, the relative content is about 36%. The compound can significantly inhibit growth of alternaria alternata mycelia, and has a significant prevention effect on tobacco brown spot. The extraction process is simple, and the product can be fully dissolved in various organic solvents. The invention provides a new resource for the creation of new pesticides and the research and development of bionic pesticides.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of agricultural prevention and treatment, and particularly relates to a Melaleuca alternifolia extract, a preparation method and application thereof in prevention and treatment of tobacco brown spot. BACKGROUND

[0002] Tobacco brown spot is a kind of fungal disease caused by Alternaria alternata, and has the characteristics of short incubation period and fast epidemic speed. The disease mainly occurs in the maturation period of tobacco leaves, and the disease spot of leaf presents concentric ring pattern, which has great influence on the appearance quality and internal quality of tobacco leaves. When the disease is serious, the yield of tobacco leaves is reduced by more than 50%. In recent years, due to the change of climate conditions and cultivation techniques, the occurrence of tobacco brown spot is increasing year by year, which brings great loss to flue-cured tobacco production. At present, the prevention and treatment of tobacco brown spot in China is still mainly based on chemical agents, and the commonly used agents include dimethachlone, polyoxin, prohexadione and azoxystrobin. However, due to the long-term use of chemical pesticides, the resistance of tobacco brown spot to the pesticides is increased, the prevention and treatment effect is greatly reduced, the "3R" problem is serious, and the quality of tobacco leaves is reduced, which is not conducive to the sustainable development of tobacco industry in China.

[0003] Melaleuca alternifolia is a shrub tree species of Myrtaceae and Melaleuca, and the leaves and tender branches thereof are rich in essential oil. The essential oil has wide antibacterial spectrum, good drug resistance and high antibacterial activity, and is effective in preventing and treating various fungi, bacteria and viruses. Meanwhile, the essential oil has less toxicity to human and animals, and does not pollute the environment, and is widely used in daily chemical, pharmaceutical and food industries.

[0004] Therefore, there is an urgent need for a Melaleuca alternifolia extract, a preparation method and application thereof in prevention and treatment of tobacco brown spot, so as to solve the problems of poor prevention and treatment effect and high cost of the chemical pesticides. SUMMARY

[0005] The present application aims at overcoming the above-mentioned deficiencies in the prior art, and providing a Melaleuca alternifolia extract, a preparation method and application thereof in prevention and treatment of tobacco brown spot.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0007] The present application provides a Melaleuca alternifolia extract in the first aspect, and the main component is pinene-4-ol, the relative content is 36%, the molecular formula is C 10 H 18 O, and the structure is as follows:

[0008]

[0009] The second aspect of the present application provides a preparation method for preparing the above-mentioned extract of Melaleuca alternifolia, comprising the following steps:

[0010] S1, collecting fresh leaves and young branches of Melaleuca alternifolia, washing and drying in the shade, and cutting into 0.5 cm in size;

[0011] S2, 100 g of the treated fresh leaves and young branches of Melaleuca alternifolia are loaded into a distillation kettle, and 500 mL of distilled water is added;

[0012] S3, heating to boiling, passing in water vapor, maintaining the temperature at 100℃, and distilling for 2 hours;

[0013] S4, the steam is cooled by a condenser, and an oil-water mixture is collected, the condensate is introduced into an oil-water separator, and the upper oil phase is separated after standing for 30 minutes;

[0014] S5, drying the oil phase with anhydrous sodium sulfate, and filtering to obtain the Melaleuca alternifolia essential oil.

[0015] The third aspect of the present application provides an application of the extract of Melaleuca alternifolia in preventing and treating tobacco brown spot.

[0016] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0017] The compound can significantly inhibit the growth of Alternaria alternata mycelium, has a significant effect on tobacco brown spot prevention, and has a simple extraction process and can be fully dissolved in various organic solvents. The present application provides a new resource for the creation of new pesticides and the development of biomimetic pesticides, and has good market application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The influence of the extract of Melaleuca alternifolia on the growth of Alternaria alternata mycelium in the present application;

[0019] Figure 2 The in vitro prevention effect of the extract of Melaleuca alternifolia on tobacco brown spot in the present application. DETAILED DESCRIPTION

[0020] The present application will be further described in detail below in combination with test examples and specific embodiments. However, it should not be understood that the scope of the above-mentioned subject matter of the present application is limited to the following examples only, and any technology realized based on the content of the present application belongs to the scope of the present application.

[0021] The first aspect of the present embodiment provides an extract of Melaleuca alternifolia, the main component of which is pinene-4-ol, the relative content of which is 36%, the molecular formula of which is C 10 H 18 O, and the structure of which is:

[0022]

[0023] The compound can significantly inhibit the mycelial growth of Alternaria alternata, has a significant effect on the prevention of tobacco brown spot, and can be fully dissolved in various organic solvents.

[0024] The second aspect of the embodiment provides a preparation method for preparing the extract of Melaleuca alternifolia.

[0025] S1, collecting fresh leaves and tender branches of Melaleuca alternifolia, washing and drying in the shade, and cutting into 0.5 cm in size;

[0026] S2, 100 g of the treated fresh leaves and tender branches of Melaleuca alternifolia are loaded into a distillation kettle, and 500 mL of distilled water is added;

[0027] S3, heating to boiling, passing in steam, maintaining the temperature at 100 DEG C, and distilling for 2 hours;

[0028] S4, the steam is cooled by a condenser, and an oil-water mixture is collected, the condensate is introduced into an oil-water separator, and the upper oil phase is separated after standing for 30 minutes;

[0029] S5, the oil phase is dried with anhydrous sodium sulfate, and Melaleuca alternifolia essential oil is obtained after filtration.

[0030] The extract is identified: the extract is diluted to 1 mg / mL with n-hexane, filtered, and then injected. GC conditions: HP-5MS capillary column (30 m x 0.25 mm x 0.25 μm); the injection port temperature is 250 DEG C; the carrier gas is high-purity helium with a flow rate of 1 mL / min; programmed temperature is used, the initial temperature is 50 DEG C, maintained for 2 minutes, increased to 250 DEG C at a rate of 10 DEG C / min, and maintained for 10 minutes. MS conditions: EI ion source, ion source temperature 230 DEG C, scan range: m / z 50-500. The chemical components in the extract are identified by comparison with the NIST mass spectrum library, and the relative content of each component is calculated (peak area normalization method).

[0031] According to the above analysis, the main component of the Melaleuca alternifolia extract is identified as Terpinen-4-ol, with a relative content of about 36%, a molecular formula of C 10 H 18 O, and a structural formula:

[0032]

[0033]

[0034] The third aspect of the embodiment provides an application of the Melaleuca alternifolia extract in the prevention and treatment of tobacco brown spot.

[0035] Example 1: Component identification analysis of Melaleuca alternifolia extract

[0036] Fresh leaves and twigs of Melaleuca alternifolia were collected, washed, and dried in the shade. The dried plant material was cut into 0.5 cm pieces. 100 g of the treated plant material was placed in a distillation kettle and 500 mL of distilled water was added. The mixture was heated to boiling, and steam was passed through the kettle to maintain a temperature of 100°C for 2 hours. The steam was cooled by a condenser, and the oil-water mixture was collected. The condensate was introduced into an oil-water separator, and the upper oil phase (crude extract) was separated after standing for 30 minutes. The oil phase was dried with anhydrous sodium sulfate, and the filtrate was obtained as the essential oil of Melaleuca alternifolia.

[0037] The extract was diluted with n-hexane to 1 mg / mL, and the sample was filtered before injection. GC conditions: HP-5MS capillary column (30 m x 0.25 mm x 0.25 μm); injection port temperature 250°C; carrier gas: high-purity helium, flow rate 1 mL / min; programmed temperature, initial temperature 50°C, hold for 2 min, increase to 250°C at 10°C / min, hold for 10 min. MS conditions: EI ion source, ion source temperature 230°C, scan range: m / z 50-500. The chemical components in the extract were identified by comparison with the NIST mass spectral library, and the relative content of each component was calculated (peak area normalization method).

[0038] According to the above analysis, the main component of the Melaleuca alternifolia extract was identified as Terpinen-4-ol, with a relative content of about 36%, a molecular formula of C 10 H 18 O, and a structure:

[0039]

[0040]

[0041] Example 2: Inhibition of Alternaria alternata mycelial growth by Melaleuca alternifolia extract

[0042] Alternaria alternata collected from 5 domestic locations was activated on PDA plates and cultured at 28°C for 3-5 days for standby use. 1, 2, 4, 8, and 10 mg / mL of culture medium containing Melaleuca alternifolia extract were prepared, and then a circular piece of the fungus (diameter 0.60 cm) was punched from the edge of the newly activated Alternaria alternata using a puncher, and the test pathogen was placed in the center of the culture medium containing different concentrations of Melaleuca alternifolia extract using a inoculation needle. The culture dishes were inverted in a 28°C incubator and the mycelial growth of the pathogen was observed in time. The inhibition effect was as follows: Figure 1The results show that the inhibition of the Melampsora thymi extract on the Alternaria alternata is concentration-dependent, the higher the concentration, the stronger the inhibition, and the highest inhibition rate is 78.14%-80.22%; and the extract has broad-spectrum resistance to Alternaria alternata in different regions. The mycelium treated with the Melampsora thymi extract and the control mycelium are observed under a microscope, and it is found that when the concentration of the Melampsora thymi extract is 8 mg / mL, the mycelium of the Alternaria alternata appears different degrees of swelling (such as Figure 1

[0043] Table 1 Broad-spectrum inhibition of the Melampsora thymi extract on the Alternaria alternata

[0044]

[0045] Example 3: In-vitro leaf piece prevention effect verification of the Melampsora thymi extract on tobacco brown spot

[0046] Healthy tobacco K326 mature leaves are selected, and after collection, the leaves are surface-disinfected with 75% ethanol for 30 s, then washed with sterile distilled water for 3 times, dried on an ultra-clean bench, wrapped with moisture-retaining absorbent cotton at the petiole, and placed in a culture dish containing a moist filter paper for inoculation. A pre-activated Alternaria alternata agar cake is taken, and the mycelium-containing side is tightly attached to one side of the tobacco leaf, and 20 μl of the Melampsora thymi extract with a concentration of 8 mg / mL is added at the inoculation site; the other side is treated with the same volume of sterile water as a control. After 7 days of 28°C moist culture, when the control side is diseased, the disease spot diameter is measured by the "cross intersection" method and the disease condition is observed. As shown in Figure 2 the in-vitro prevention effect test results show that the control mycelium has been obviously diseased after 7 days, and the mycelium penetrates the leaf and grows on the back, and the leaf at the inoculation site shows symptoms such as yellowing and wilting; while the leaf treated with the Melampsora thymi extract with a concentration of 8 mg / mL is green, and no mycelium grows on the back. This shows that the Melampsora thymi extract with a concentration of 8 mg / mL can reduce the disease degree of tobacco brown spot to a certain extent, and the in-vitro prevention effect is 71.2%, so the Melampsora thymi extract has a certain application potential.

[0047] Example 4: Potting prevention effect verification of the Melampsora thymi extract on tobacco brown spot

[0048] ​After tobacco transplanting, when tobacco seedlings grow to 8 true leaves, 8 mg / mL of the extract of Melaleuca alternifolia is uniformly sprayed on tobacco leaves, with the control group being sprayed with water and 40% dimethachlone 500 times dilution liquid respectively, 15 tobacco seedlings per treatment, 24 h later, tobacco brown spot spores are inoculated by hanging drop method, 6 drops per leaf, 20 ul per drop, 25℃ moist culture for 7 d, disease index and control effect are calculated. The results show that 8 mg / mL of the extract of Melaleuca alternifolia can effectively reduce the disease index of tobacco brown spot, and the control effect on tobacco brown spot is 65.3%, which is increased by 34.4% compared with the control effect (45.8%) of 40% dimethachlone 500 times dilution liquid.

[0049] Table 2 potting control effect of the extract of Melaleuca alternifolia on tobacco brown spot

[0050]

[0051] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement and improvement within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Melaleuca alternifolia extract, characterized in that The main component is terpinen-4-ol, with a relative content of 36% and a molecular formula of C 10 H 18 O, the structure is:

2. A preparation method for preparing the Melaleuca alternifolia extract according to claim 1, characterized in that: The steps include: S1. Collect fresh leaves and twigs of Melaleuca alternifolia, wash and dry in the shade, and chop into 0.5 cm pieces; S2, the fresh leaves and shoots of Melaleuca alternifolia after 100g treatment are loaded into a still, and 500mL of distilled water is added; S3, heating to boiling, introducing water vapor, maintaining the temperature at 100°C, and distilling for 2 hours; S4, the steam is cooled through a condenser, the oil-water mixture is collected, and the condensate is introduced into an oil-water separator, left to stand for 30 minutes, and the upper oil phase is separated; S5. Dry the oil phase with anhydrous sodium sulfate, and filter to obtain the Melaleuca alternifolia essential oil.

3. Application of a Melaleuca alternifolia extract in preventing and treating tobacco brown spot disease.

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