CN104782669B - A kind of Extracts from Plant Recourses for preventing and treating tobacco ralstonia solanacearum - Google Patents
A kind of Extracts from Plant Recourses for preventing and treating tobacco ralstonia solanacearum Download PDFInfo
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- CN104782669B CN104782669B CN201510210103.5A CN201510210103A CN104782669B CN 104782669 B CN104782669 B CN 104782669B CN 201510210103 A CN201510210103 A CN 201510210103A CN 104782669 B CN104782669 B CN 104782669B
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Abstract
A kind of Extracts from Plant Recourses for preventing and treating tobacco ralstonia solanacearum, this extract is ethyl acetate extract or the water extract of dry Herba Capsellae, this Herba Capsellae ethyl acetate extract and water extract are respectively provided with inhibitory action to tobacco ralstonia solanacearum, wherein the inhibition of ethyl acetate extract is best, its suppression ratio is 56.9%, using ethyl acetate to extract the active matter in Herba Capsellae root, stem, seed, leaf respectively, find that the fungistatic effect of root and leaf is the strongest, the fungistatic effect of seed and stem extract is more weak；Meanwhile, the result of study of the present invention also shows, the fungistatic effect of dry Herba Capsellae extract is apparently higher than fresh Herba Capsellae extract.The inhibitory action that tobacco ralstonia solanacearum is had by the Herba Capsellae extract of the present invention shows that Herba Capsellae has the New function of controlling plant diseases, and the biological pesticide new for exploitation Herba Capsellae natural active matter and research and development provides scientific basis.
Description
Technical field
The invention belongs to botanical pesticide technical field, be specifically related to a kind of plant source for preventing and treating tobacco ralstonia solanacearum
Extract.
Technical background
Herba Capsellae (Capsella bursa-pastoris) belongs to Cruciferae Herba Capsellae and belongs to herbaceous plant, and meanwhile, Herba Capsellae also can enter
Medicine, is also medicinal and edible plant.Herba Capsellae is distributed mainly on subtropical zone and Temperate Region in China, and China all can grow from south to north, especially
The most common at limes marginis, ridge, roadside etc..
Herba Capsellae is multiple rich in organic acid, Fumaric acid, polypeptide, tannin, flavonoid, alkaloid, aminoacid, ergocristine etc.
Active component, has analgesia, spasmolytic, improving eyesight, strengthening the tendons and bones, blood pressure lowering, antiinflammatory, antibacterial, anticancer, arrhythmia, lipotropism matter mistake
Oxidation, antiviral, health care are waited for a long time and are prevented disease and effect for the treatment of disease.Although the research of the medical aspect of Herba Capsellae is more, but sharp
Rarely seen by Herba Capsellae research report in terms of preventing and treating plant pathogenetic bacteria disease.Therefore, the present invention is thin to pathogenic with Herba Capsellae
Bacterium this problem in science the most inhibited, with tobacco ralstonia solanacearum as indicator bacteria, explores the extraction side of Herba Capsellae bateriostatics
Method, specifies different parts and produces the amount of antibacterial substance, extraction efficiency and MIC value, the potentiality to be exploited of assessment Herba Capsellae, it is intended to for from now on
Develop botanical pesticide and theoretical foundation and practical basis are provided.
Summary of the invention
The technical problem to be solved is: for the technical blank of Herba Capsellae controlling plant diseases, it is provided that one
Plant the Extracts from Plant Recourses for preventing and treating tobacco ralstonia solanacearum.
In order to solve above-mentioned technical problem, the technical solution adopted in the present invention is: one is used for preventing and treating tobacco bacterial wilt
The Extracts from Plant Recourses of bacterium, is characterized in, described Extracts from Plant Recourses is Herba Capsellae extract.
Above-mentioned Herba Capsellae extract is ethyl acetate extract or the water extract of Herba Capsellae.
Above-mentioned Herba Capsellae extract is to take fresh Herba Capsellae to clean, and drying to water content is 0.5-3.5%, pulverizes, uses acetic acid second
Herba Capsellae powder is extracted by ester or sterilized water, and gained filtrate enters Rotary Evaporators and is concentrated into paste and obtains.Wherein, ethyl acetate is used
Or Herba Capsellae powder is extracted the ratio referring to add 20-25mL ethyl acetate or sterilized water in 1g Herba Capsellae powder, in Herba Capsellae by sterilized water
Powder adds ethyl acetate or sterilized water, shakes 24-48h in 25-26 DEG C with 80-120r/min, filter, collect filtrate, filtering residue
Repeat to extract 1 time, merge twice filtrate.The pulverizing of indication is that to be crushed to 100-120 mesh with the rotating speed of 8000-12000r/min thin
Powder.Described rotary evaporation condition: pressure is 0.09-0.1MPa, bath temperature is 60 DEG C, and condensation temperature is 6-8 DEG C, and rotating speed is
80-110r/min。
The Herba Capsellae extract (ethyl acetate extract or water extract) of the present invention has suppression and makees tobacco ralstonia solanacearum
With, show that Herba Capsellae has the New function of controlling plant diseases；The realization of the present invention simultaneously, for developing Herba Capsellae natural active products
Matter and research and development are prepared neoplasm pesticide and are provided scientific basis.
Accompanying drawing explanation
Fig. 1 is the ethyl acetate extract inhibitory action to tobacco ralstonia solanacearum of Herba Capsellae.
Wherein: A represents the ethyl acetate extract of Herba Capsellae；B represents ethyl acetate.
Fig. 2 is the water extract inhibitory action to tobacco ralstonia solanacearum of Herba Capsellae.
Wherein: A represents the water extract of Herba Capsellae；B represents sterilized water.
Detailed description of the invention
Test plant and bacterial strain
Fresh Herba Capsellae is from scientific base, Agricultural University Of Hunan Liuyang；Tobacco ralstonia solanacearum (Ralstonia
Solanacearum), bio-safety inspection center of Agricultural University Of Hunan II provide.
The activation of tobacco ralstonia solanacearum strain: use inoculating loop to scrape 3 ring strains and access beef extract-peptone slant culture
Base (NA), cultivates 24-48h continuously at 26-28 DEG C, after bacterium colony grows, make concentration 1.1 × 108The bacteria suspension of CFU/mL (with
Lower abbreviation bacteria suspension), standby.
For examination culture medium
Beef-protein medium (NA): Carnis Bovis seu Bubali cream 5g, peptone 8g, sodium chloride 5g, agar 20g, distilled water
1000mL、pH 7.0-7.2；
Embodiment 1 Herba Capsellae ethyl acetate extract and the water extract inhibitory action to tobacco ralstonia solanacearum
Take fresh Herba Capsellae (including root, stem, leaf, seed) clean, dry to water content be 2.5%, with 8000-12000r/min
Rotating speed be crushed to 100-120 mesh fine powder.Weigh quantitative Herba Capsellae dry powder sample, add 500mL ethyl acetate, in 25-26 DEG C
Shaking 36h with 100r/min, filter, collect filtrate, filtering residue repeats to extract 1 time, merges twice filtrate, filtrate is placed in pressure and is
0.09-0.1Mpa, bath temperature is 60 DEG C, and condensation temperature is 6 DEG C, rotating speed be 80r/min Rotary Evaporators in be concentrated into cream
Shape, obtains ethyl acetate extract.With ethyl acetate constant volume ethyl acetate extract, 4 DEG C of preservations, standby.
According to said method, preparing the water extract of Herba Capsellae with sterilized water for Extraction solvent, water extract uses sterilized water fixed
Hold.
Using inhibition zone method to measure the Herba Capsellae extract fungistatic effect to tobacco ralstonia solanacearum, the NA culture medium of sterilizing is poured into
Treating in culture dish that it cools down, liquid-transfering gun pipettes 200 μ L bacteria suspensions, and uniformly, card punch (7mm) punches in culture medium in coating, note
Enter 100 μ L ethyl acetate or sterilized water extraction things, with ethyl acetate or sterilized water for comparison, be repeated 3 times, plate be placed in respectively
Being placed in 26-28 DEG C of bacteriological incubator and cultivate 24h, observed result, decussation method measures antibacterial circle diameter, calculates suppression ratio, knot
Fruit see table 1.
In conjunction with seeing Fig. 1 and Fig. 2, result shows: Herba Capsellae ethyl acetate and sterilized water extraction thing are equal to tobacco ralstonia solanacearum
Inhibited, wherein the extraction effect of ethyl acetate is best, and its antibacterial circle diameter is 36.5mm, and suppression ratio is 56.9%,
The inhibition of sterilized water extraction thing is taken second place, and its antibacterial circle diameter is 17.8mm, and suppression ratio is 28.3%.As can be seen here, use
It is effective that ethyl acetate extracts Herba Capsellae bateriostatics, can extract antipathogenic composition to greatest extent.
The impact on tobacco ralstonia solanacearum inhibitory action of table 1 different solvents
| Fungistatic effect | Ethyl acetate extract | Sterilized water extraction thing |
| Antibacterial circle diameter (mm) | 36.5±0.7638 | 17.8±0.2887 |
| Suppression ratio (%) | 56.9Aa±1.1676 | 28.3Bb±0.4619 |
Note: different lower cases represent significant difference (ρ < 0.05), different capitalizations represent pole significant difference (ρ <
0.01)。
The different parts extract of the embodiment 2 Herba Capsellae inhibitory action to tobacco ralstonia solanacearum
Taking fresh Herba Capsellae root to clean, drying to water content is 2.5%, is crushed to 100-120 with the rotating speed of 10000r/min
Mesh fine powder.Weigh quantitative Herba Capsellae root dry powder, add 400mL ethyl acetate, shaking table concussion 36h, collect filtrate；Filtering residue repeats to carry
Take once, merge 2 filtrate, enter Rotary Evaporators and be concentrated to give paste, it is thus achieved that the extract of Herba Capsellae root, fixed by ethyl acetate
Hold to 20mL.According to method of the same race, prepare Herba Capsellae leaf, stem, seed ethyl acetate extract respectively.
Inhibition zone method is used to measure the Herba Capsellae different parts extract fungistatic effect to tobacco ralstonia solanacearum, the NA training of sterilizing
Foster base is poured into and is treated in culture dish that it cools down, and liquid-transfering gun pipettes 200 μ L bacteria suspensions, and uniformly, card punch (7mm) is in culture medium in coating
Punching, injects 100 μ L extracts, and ethyl acetate compares, and is repeated 3 times, and is placed in by plate and is respectively placed in 26-28 DEG C of antibacterial culturing
Case cultivates 24h, observed result, and decussation method measures antibacterial circle diameter, calculates suppression ratio, and result see table 2.
Result shows: Herba Capsellae root, stem, leaf, the ethyl acetate extract of seed all have inhibitory action to tobacco ralstonia solanacearum, its
Suppression ratio is respectively 66.7%, 57.4%, 66.9%, 62.7%, Qi Zhonggen, leaf the fungistatic effect of extract best, both
Suppression ratio there was no significant difference；The fungistatic effect of seed extract takes second place；The fungistatic effect of stem extract is the most worst；Root and leaf, stem,
There is mutually pole significant difference in the triangular suppression ratio of seed.As can be seen here, the root of Herba Capsellae, stem, leaf, seed there is antibacterial substance
Producing, its extract part is different, and fungistatic effect is the most different.
The impact on tobacco ralstonia solanacearum inhibition of the table 2 Herba Capsellae different parts ethyl acetate extract
Note: different lower cases represent that significant difference (ρ < 0.05), different capitalizations represent pole significant difference.
The extraction efficiency of embodiment 3 Herba Capsellae different parts extract
Taking fresh Herba Capsellae root to clean, drying to water content is 2.5%, is crushed to 100-120 with the rotating speed of 10000r/min
Mesh fine powder.Weigh 20g Herba Capsellae root powder, according to the method in embodiment 2, with ethyl acetate as Extraction solvent, prepare Herba Capsellae root and extract
Thing paste.Utilize minusing to calculate paste weight, calculate Herba Capsellae root extract according to dry powder quality and paste quality
Extraction ratio.Obtain the ethyl acetate extract paste of Herba Capsellae stem, leaf, seed according to said extracted method respectively, calculate and extract
Rate, result see table 3.
Result shows: the extraction ratio of Herba Capsellae leaf is up to 15.2%, and the extraction ratio of Herba Capsellae seed takes second place, and Herba Capsellae stem and root
Extraction ratio is consistently lower than other positions.Illustrate the root of Herba Capsellae, stem, leaf, seed all contain antibacterial substance, but the how rare difference of its content
Not, wherein in Herba Capsellae leaf, antibacterial substance is more, takes second place in seed, and stem and root are relatively low.
The extraction ratio of table 3 Herba Capsellae different parts ethyl acetate extract
| Fungistatic effect | Root | Stem | Leaf | Seed |
| Dry powder quality (g) | 20.0 | 20.0 | 20.0 | 20.0 |
| Paste quality (g) | 2.1 | 2.6 | 3.0 | 2.9 |
| Extraction ratio (%) | 10.5% | 13.1% | 15.2% | 14.5% |
The embodiment 4 Herba Capsellae different parts extract MIC value to tobacco ralstonia solanacearum
Dissolve the ethyl acetate extraction paste of Herba Capsellae root, stem, leaf, seed respectively with dehydrated alcohol, being configured to concentration is
The mother solution of 80mg/mL, is diluted to 6 kinds of different gradient concentration (40mg/mL, 20mg/mL, 10mg/ with doubling dilution by mother solution
ML, 5mg/mL, 2.5mg/mL, 1.25mg/mL), measure the root of 7 kinds of variable concentrations respectively with Beating holes method, stem, leaf, seed carry
Taking the thing fungistatic effect to tobacco ralstonia solanacearum, dehydrated alcohol compares, and every concentration is repeated 3 times, with carrying of integral asepsis growth
Taking the minimum inhibitory concentration that thing highest dilution is this extract, result see table 4.
Result shows: when between the concentration of 4 kinds of extracts is at 5mg/mL to 80mg/mL, they are to tobacco ralstonia solanacearum
Growth all produce inhibitory action；When concentration is 2.5mg/mL, the only extract of root has a bacteriostasis, stem, leaf, the carrying of seed
Take thing and there is no bacteriostasis；But when concentration is reduced to 1.25mg/mL, 4 kinds of extracts are all without bacteriostasis.Therefore, root,
Stem, leaf, the MIC of seed ethyl acetate extract are respectively 2.5mg/mL, 5mg/mL, 5mg/mL, 5mg/mL.
The MIC value of table 4 Herba Capsellae different parts ethyl acetate extract
| Extract part | Concentration (mg/mL) |
| 80 | 40 | 20 | 10 | 5 | 2.5 | 1.25 | |
| Root | + | + | + | + | + | + | - |
| Stem | + | + | + | + | + | - | - |
| Leaf | + | + | + | + | + | - | - |
| Seed | + | + | + | + | + | - | - |
Note: "+" there is fungistatic effect, "-" is without fungistatic effect.
Embodiment 5 Different Extraction Method inhibitory action to tobacco ralstonia solanacearum
The preparation of fresh Herba Capsellae extract: take quantitative fresh Herba Capsellae (including root, stem, leaf and seed) and clean, dry surface
Moisture, shreds with shears and inserts in mortar, and in 15-25 DEG C of grinding, proceeding to volume is 1000mL triangular flask, adds 500mL acetic acid
Ethyl ester, under the conditions of 25-26 DEG C, 100r/min shakes 368h, filters, and collects filtrate；Filtering residue repeats to extract 1 time, merges twice
Filtrate, obtains the thick juice of Herba Capsellae through double gauze and filter paper filtering, then it is higher to filter acquisition purity through 0.22 μm biofilter
Herba Capsellae juice.Herba Capsellae juice is placed in pressure be 0.09-0.1MPa, bath temperature be 60 DEG C, condensation temperature be 6-8 DEG C, turn
The Rotary Evaporators that speed is 80-110r/min is concentrated into paste, and sterilized water dissolves constant volume, and 4 DEG C save backup.
The preparation of dry Herba Capsellae extract: take fresh Herba Capsellae (including root, stem, leaf and seed) and clean, dries to water content and is
2.5%, it is crushed to 100-120 mesh fine powder with the rotating speed of 10000r/min.Weigh quantitative Herba Capsellae dry powder sample, add 500mL
Ethyl acetate, under the conditions of 25-26 DEG C, 100r/min shakes 368h, filters, and collects filtrate；Filtering residue repeats to extract 1 time, merges two
Secondary filtrate, being placed in pressure is 0.09-0.1Mpa, and bath temperature is 60 DEG C, and condensation temperature is 6 DEG C, and rotating speed is the rotation of 80r/min
Evaporimeter is concentrated into paste, and sterilized water dissolves constant volume, and 4 DEG C save backup.
Inhibition zone method is used to measure the ethyl acetate extract of the Herba Capsellae fungistatic effect to tobacco ralstonia solanacearum, the NA of sterilizing
Culture medium is poured into and is treated in culture dish that it cools down, and liquid-transfering gun pipettes 200 μ L bacteria suspensions, and uniformly, card punch (7mm) is in culture medium in coating
Upper punching, injects 100 μ L sterilized water or ethyl acetate extracts, with sterilized water or ethyl acetate for comparison, is repeated 3 times, will be flat
Ware is placed in and is respectively placed in 26-28 DEG C of bacteriological incubator cultivation 24h, observed result, and decussation method is measured antibacterial circle diameter, calculated
Suppression ratio, result see table 5.
As shown in Table 5, dry Herba Capsellae ethyl acetate extract is higher than fresh Herba Capsellae to the inhibition of tobacco ralstonia solanacearum
Ethyl acetate extract, its antibacterial circle diameter and suppression ratio are 1.3 times of dry Herba Capsellae extract respectively, 1.36 times；Variance analysis
Result also indicates that dry Herba Capsellae ethyl acetate extract and the fresh Herba Capsellae extract inhibition to tobacco ralstonia solanacearum, the poorest
Different significantly, and difference is the most notable；Illustrating that the content of dry Herba Capsellae bateriostatics is higher than fresh Herba Capsellae, this may be with fresh Herba Capsellae moisture
Content height is relevant.
The impact on tobacco ralstonia solanacearum inhibitory action of table 5 Different Extraction Method
| Fungistatic effect | Dry Herba Capsellae ethyl acetate extract | Fresh Herba Capsellae ethyl acetate extract |
| Antibacterial circle diameter (mm) | 32.33±0.4522 | 24.8±0.5647 |
| Suppression ratio (%) | 52.36Aa±0.7431 | 38.45Bb±0.3211 |
Note: different lower cases represent significant difference (ρ < 0.05), different capitalizations represent pole significant difference (ρ <
0.01)。
Claims (6)
1. the Extracts from Plant Recourses application in preventing and treating tobacco ralstonia solanacearum, it is characterised in that described Extracts from Plant Recourses
For ethyl acetate extract or the water extract of Herba Capsellae, be to take fresh Herba Capsellae to clean, dry to water content be 0.5-3.5%, powder
Broken, with ethyl acetate or sterilized water, Herba Capsellae powder to be extracted, gained filtrate enters Rotary Evaporators and is concentrated into paste and obtains.
Apply the most as claimed in claim 1, it is characterised in that Herba Capsellae powder is extracted by described ethyl acetate or sterilized water
Refer to add in 1 gram of Herba Capsellae powder the ratio of 20-25mL ethyl acetate or sterilized water, in Herba Capsellae powder, add ethyl acetate or aseptic
Water, shakes 24-48h in 25-26 DEG C with 80-120r/min, filters, and collects filtrate, and filtering residue repeats to extract 1 time, merges twice filter
Liquid.
Apply the most as claimed in claim 2, it is characterised in that described pulverizing is to pulverize with the rotating speed of 8000-12000r/min
To 100-120 mesh fine powder.
4. the Extracts from Plant Recourses application in preparation preventing and treating tobacco bacterial wilt biological pesticide, it is characterised in that described in plant
Thing source extract refers to ethyl acetate extract or the water extract of Herba Capsellae, is to take fresh Herba Capsellae to clean, and dries to water content and is
0.5-3.5%, pulverizes, extracts Herba Capsellae powder with ethyl acetate or sterilized water, and gained filtrate enters Rotary Evaporators and is concentrated into
Paste obtains.
Apply the most as claimed in claim 4, it is characterised in that Herba Capsellae powder is extracted by described ethyl acetate or sterilized water
Refer to add in 1 gram of Herba Capsellae powder the ratio of 20-25mL ethyl acetate or sterilized water, in Herba Capsellae powder, add ethyl acetate or aseptic
Water, shakes 24-48h in 25-26 DEG C with 80-120r/min, filters, and collects filtrate, and filtering residue repeats to extract 1 time, merges twice filter
Liquid.
Apply the most as claimed in claim 5, it is characterised in that described pulverizing is to pulverize with the rotating speed of 8000-12000r/min
To 100-120 mesh fine powder.
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Inventor after: Liu Shuangqing Inventor after: Zhang Ya Inventor after: Liao Xiaolan Inventor after: Xie Yan Inventor before: Zhang Ya Inventor before: Liu Shuangqing Inventor before: Liao Xiaolan Inventor before: Xie Yan |
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Application publication date: 20150722 Assignee: Hunan Hewang Biotechnology Co.,Ltd. Assignor: Hunan Agricultural University Contract record no.: X2022980024840 Denomination of invention: A kind of plant extract for controlling tobacco bacterial wilt Granted publication date: 20161005 License type: Common License Record date: 20221206 |