CN110786341A - Preparation method and application of lotus plumule extracting solution - Google Patents
Preparation method and application of lotus plumule extracting solution Download PDFInfo
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- CN110786341A CN110786341A CN201911120874.XA CN201911120874A CN110786341A CN 110786341 A CN110786341 A CN 110786341A CN 201911120874 A CN201911120874 A CN 201911120874A CN 110786341 A CN110786341 A CN 110786341A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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Abstract
The invention relates to the field of biology, and particularly relates to a preparation method and application of a lotus plumule extracting solution. The lotus plumule extract can effectively inhibit the rice bacterial leaf blight, induce the expression of rice defense genes and increase the basic resistance of rice.
Description
Technical Field
The invention relates to the field of biology, and particularly relates to a preparation method and application of a lotus plumule extracting solution.
Background
Bacterial blight of rice is caused by Xanthomonas oryzae pv. oryzae, one of the major diseases of rice in china. Pathogenic bacteria of rice bacterial leaf blight comprise two types of bacterial leaf blight bacteria and alternaria alternate, the bacterial leaf blight bacteria are gram-negative bacteria, have no spore and capsule, and are surrounded by exosporium polysaccharide with mucilage outside the bacteria. At present, the prevention and treatment of the bacterial blight are mainly chemical agents, but the drug effect is poor and certain pollution is brought when the bacterial blight is seriously attacked. The method is sometimes generated in each rice production area in China, and damages to withering of rice leaves and reduction of yield cause serious threat to stable yield of rice. At present, the main way for preventing and treating rice bacterial blight is chemical prevention and treatment, and the rice bacterial blight is prevented and treated by using chemical preparations such as streptomycin, chloramphenicol, bismerthiazol and thifluzole. Although the composition plays a certain role in preventing and treating the rice bacterial blight at the initial stage of the disease, the prevention and treatment effect is poor when the disease is serious, and great negative effects are also generated. The excessive use of chemical pesticides easily causes environmental pollution, damages the physicochemical property of soil and ecological balance, and leads to a series of problems of enhancing the resistance of pathogenic bacteria and unsafe chemical pesticide residue on human bodies. The biological control has the advantages of safety and no pollution to the environment, and is an effective way for controlling rice bacterial blight. Research shows that the plant contains a great amount of bacteriostatic active substances and has broad-spectrum bacteriostatic property. The Wuling mountain area has wide regions, unique climatic conditions and rich plant resources, and is a resource treasury in China.
Disclosure of Invention
The invention aims to provide a lotus plumule extracting solution capable of inhibiting rice bacterial blight and a preparation method thereof.
The technical scheme provided by the invention is that the lotus plumule extracting solution is prepared by the following steps:
step 1: drying the lotus plumule, and grinding into powder;
step 2: dissolving the powder in the step 1 by using ethanol for the first time, performing ultrasonic treatment and centrifugation, and taking supernate;
and step 3: drying the supernatant obtained in the step 2 under reduced pressure, and dissolving with distilled water;
and 4, step 4: and (4) extracting the solution obtained in the step (3) by using chloroform, drying under reduced pressure again, and then adding ethanol for secondary dissolution to obtain the lotus plumule extracting solution.
And in the step 2, the first dissolution with ethanol is to mix the powdery lotus plumule with 90% ethanol according to a feed-liquid ratio of 1: 15 are dissolved.
Furthermore, the ethanol added in step 4 is ethanol added at a concentration of 90%.
And the lotus plumule extract has the inhibiting effect on rice bacterial blight.
And the lotus plumule extract has the induction effect on rice defense gene expression.
Moreover, the rice defense genes are WRKY45 gene, PR1b gene and PZ1 gene.
Compared with the prior art, the technical scheme has the beneficial effects that: the lotus plumule extract can effectively inhibit rice bacterial leaf blight and induce rice defense gene expression. The extract is a botanical fungicide which is safe to human and livestock, has no pollution, low toxicity and low residue.
Drawings
FIG. 1 is a histogram of mean diameter of zone of inhibition of plant extracts;
FIG. 2 is a diagram of the bacteriostatic effect of plumula Nelumbinis (n-butanol);
FIG. 3 is a diagram of the bacteriostatic effect of plumula Nelumbinis (petroleum ether);
FIG. 4 is a diagram of bacteriostatic effect of plumula Nelumbinis (chloroform);
FIG. 5 shows the results of in vivo prevention and treatment of plumula Nelumbinis (petroleum ether);
FIG. 6 shows the detection of the expression level of the defense gene.
Detailed Description
The preparation method of the lotus plumule extracting solution comprises the following steps:
paddy rice bacterial blight (Xanthomonas campestris pv. oryzae (Ishiyama) Dye: supplied by the university of the southern and Central university institute of Life sciences.
The test plant extract:
safflower (water part), safflower (n-butanol), asiatic moonseed rhizome (water part), dandelion (ethyl acetate), phellodendron (ethyl acetate), lotus plumule (n-butanol), lotus plumule (petroleum ether), lotus plumule (chloroform), balsam pear (n-butanol), balsam pear (petroleum ether), balsam pear (chloroform), swertia japonica (n-butanol) and swertia japonica (ethyl acetate).
Test reagents:
ampicillin sodium (2mg/ml), 75% ethanol, hypochondrium philosophy culture medium PDA (potato 300g/L is boiled into mud shape, 6 layers of gauze are used for filtering, sucrose 15g/L, peptone 5g/L, Ca (NO3) 2.4H 2O 0.72g/L, NaH2PO 4.2H 2O2.6g/L, agar powder 20g/L, and sterile water). Trizol; axyrep total RNA miniprep kit; real-time fluorescence quantification Kit SYBRPremix Ex Taq, reverse transcription Kit PrimeScript RT reagent Kit with gDNA Eraser.
Firstly, preparing a plant extracting solution:
balsam pear (n-butanol): weighing balsam pear powder, namely weighing 90% ethanol according to a material-liquid ratio of 1: 20, dissolving; carrying out ultrasonic treatment at 56 ℃ for 1h in an ultrasonic oscillator, centrifuging at 4 ℃ and 4000rpm for 20 minutes, taking supernatant, putting the supernatant into a round-bottom flask, drying at 56 ℃ under reduced pressure in a rotary reduced-pressure drying instrument, fully dissolving the supernatant with distilled water, extracting the solution with a proper amount of n-butyl alcohol for 2-3 times, transferring the solution into a new round-bottom flask, drying the solution at 70 ℃ under reduced pressure again, and dissolving the solution with 75% ethanol to obtain a balsam pear (n-butyl alcohol) solution with a certain concentration [ 7.9 ].
Plumula Nelumbinis (chloroform): drying lotus plumule, weighing 5g of lotus plumule, grinding the lotus plumule into powder by using liquid nitrogen in a mortar, and mixing the powder with 90% ethanol in a material-liquid ratio of 1: 15, dissolving, performing ultrasonic treatment at 60 ℃ in an ultrasonic oscillator for 1h, centrifuging at 4 ℃ and 4000rpm for 20 minutes, transferring supernatant into a new round-bottom flask, drying at 60 ℃ under reduced pressure in a rotary reduced-pressure drying instrument, dissolving with distilled water for multiple times after drying, extracting with appropriate amount of chloroform for 2-3 times, transferring into the new round-bottom flask, drying at 60 ℃ under reduced pressure again, and dissolving with 5ml of 75% ethanol to obtain 1g/ml lotus plumule (chloroform) solution.
Secondly, strain activation
In a clean bench, 10ul of the bacterial strain of the bacterial blight of the bacterial leucovorin is sucked and streaked in a hucho taimen culture medium, the culture is carried out for 3 days at 28 ℃, then a single bacterial colony of the bacterial blight of the bacterial leucovorin is picked up and cultured in a hucho taimen liquid culture medium at 150rpm and 28 ℃ for 48 hours. Third, the Minimum Inhibitory Concentration (MIC) of different plant extracts to the bacterial blight of the white leaf blight
Filtering the plant extractive solution for sterilization, diluting with sterile water to different concentration gradients, sucking l ml of plant extractive solution, adding into 9ml of hot-melt PDA culture medium, mixing, pouring into a glass culture dish with diameter of 9cm, and making into culture medium with drug, with 75% ethanol as control group. Each treatment was set to 3 replicates. After the culture medium is cooled and solidified, 0.1ml of test bacterium liquid (OD value 0.6) is sucked and added to the surface of the culture medium with the medicine, and the test bacterium liquid is uniformly coated. Culturing for 48h in a constant temperature incubator at 28 ℃, observing the growth condition of pathogenic bacteria in each culture dish, and recording the result. The lowest concentration of the liquid medicine without bacteria growth is the lowest inhibitory concentration.
The results were analyzed as follows:
from table 1, it can be seen that the MIC values of lotus plumule (petroleum ether) and bitter gourd (ethyl acetate) are very small, and the bacteriostatic effect is better.
TABLE 1 MIC values of plant extracts
| Drug delivery reagent 100mg/ml | MIC(mg/ml) |
| Lotus plumule (Petroleum ether) | 1～3 |
| Balsam pear (acetic ether) | 4～5 |
| Balsam pear (n-butanol) | >5 |
| Root of Paris polyphylla | >5 |
| Rhizoma Menispermi (Water part) | >5 |
| Safflower (Water part) | >5 |
| Cortex Phellodendri (Water part) | >5 |
| Safflower (n-butyl alcohol) | >5 |
| Swertia (n-butanol) | >5 |
Fourth, bacteriostatic activity agar sheet method of plant extract
0.2ml of bacterial-blight suspension with OD value of about 0.6 is absorbed, the suspension is evenly coated on a PDA flat plate, sterilized filter paper pieces are taken out by using sterilized tweezers and evenly pasted on the flat plate, the medicament names are correspondingly marked behind the flat plate, then 5ul of ampicillin sodium (2mg/ml) is sequentially absorbed and vertically dropped on the filter paper pieces to be used as positive control, 10ul of 75% ethanol is used as negative control, plant extract with different concentration gradients is absorbed and used as experimental groups on the filter paper pieces, a sealing film is sealed in a 28 ℃ incubator to be inversely cultured for 48 hours, the diameter of a bacteriostatic ring is measured by using a ruler, and the diameter of the bacteriostatic ring is used as a bacteriostatic activity index for measuring extract phases of different plant extract.
The results were analyzed as follows:
table 2 shows the diameter mm of the zone of inhibition of plant extract
| Reagent for testing | Diameter mm of bacteriostatic zone |
| Ampicillin sodium (2mg/ml) | 10.70 |
| 75% ethanol | 9.30 |
| Lotus plumule (Petroleum ether) | 13.83 |
| Lotus plumule (n-butanol) | 13.00 |
| Lotus plumule (chloroform) | 11.75 |
| Balsam pear (n-butanol) | 9.66 |
| Balsam pear (chloroform) | 11.25 |
| Safflower (Water part) | 7.83 |
| Safflower (n-butyl alcohol) | 7.33 |
| Swertia (n-butanol) | 9.16 |
| Swertia (ethyl acetate) | 11.00 |
| Dandelion (Ethyl acetate) | 9.00 |
| Cortex Phellodendri (ethyl acetate) | 7.00 |
| Rhizoma Menispermi (Water part) | 7.83 |
The diameter of the zone of inhibition is shown in the figure and analyzed as follows:
FIG. 1 is a histogram of mean diameter of zone of inhibition of plant extracts; as can be seen from FIG. 1, the lotus plumule (petroleum ether) and the lotus plumule (n-butanol) have larger diameter of inhibition zone, namely the 13 plant extract has the best inhibition effect on the bacterial blight of white leaf blight; and lotus plumule (chloroform), balsam pear (chloroform) and swertia herb (ethyl acetate) in turn.
FIG. 2 is a diagram of the bacteriostatic effect of plumula Nelumbinis (n-butanol);
which comprises the following steps: 2-1: Positive control: 2mM Amp 5 ul;
2-2 negative control: 10ul of 75% ethanol;
2-3, experimental group: lotus seed (n-butanol) 20mg/ml 10 ul;
2-4, experimental group: lotus plumule (n-butanol) 100mg/ml 10 ul.
FIG. 3 is a diagram of the bacteriostatic effect of plumula Nelumbinis (petroleum ether);
which comprises the following steps: 3-1: positive control 2mM Amp 5 ul;
3-2 negative control: 10ul of 75% ethanol;
3-3, experimental group: lotus plumule (petroleum ether) 10ul at 1 g/ml;
3-4: experimental groups: lotus plumule (petroleum ether) 0.5g/ml 10 ul.
Fig. 4 is a bacteriostatic effect diagram of lotus plumule (chloroform), which comprises:
4-1: Positive control: 2mM Amp 5 ul;
4-2 negative control: 10ul of 75% ethanol;
4-3, experimental group: lotus plumule (chloroform) 10ul at 20 mg/ml;
4-4: experimental group: lotus plumule (chloroform) 100mg/ml 10 ul.
Five, in-vivo prevention and treatment method of plant extract on bacterial blight
The method comprises the steps of sprouting seeds of Nipponbare seeds of a rice variety and transplanting the seeds in 4 pots, setting 2 control groups and 2 experimental groups, wherein the control group 1 is not treated, the control group 2 is inoculated, the experimental group 1 is inoculated firstly and then sprayed with pesticide, and the experimental group 2 is sprayed with pesticide firstly and then inoculated. When the rice grows to 12-leaf stage, 200ml of lotus plumule (petroleum ether) extracting solution is prepared, 100ml of lotus plumule (petroleum ether) extracting solution is sprayed on the rice of the experimental group 2 on the first day, 100ml of rhizoctonia solani suspension with an OD value of about 0.6 is sucked on the second day, the bacterial blight on the control group 2, the experimental group 1 and the experimental group 2 is inoculated, and 100ml of lotus plumule (petroleum ether) extracting solution is sprayed on the rice of the experimental group 1 on the third day. Statistics is carried out after two weeks, and the length and the area of the leaf scab are measured.
The results were analyzed as follows: FIG. 5 shows the results of in vivo control of plumula Nelumbinis (petroleum ether), the average length of lesion of bacterial leaf blight infected by the sprayed leaf after inoculation is significantly reduced, which shows that plumula Nelumbinis (petroleum ether) has good inhibitory activity to bacterial leaf blight in vitro and in vivo level, and has potential research and application values.
Sixth, expression detection method of defense gene
After measuring the length and the area of the leaf lesion, respectively taking the leaf from a control group and an experimental group, extracting RNA, inverting cDNA, then analyzing the expression quantity, and respectively detecting the expression quantity of defense genes WRKY45, PZ1 and PR1 b.
The results were analyzed as follows:
in the experiment of living body prevention and control of rice inoculated with bacterial blight of the leaf blight of the rice, the lotus plumule (petroleum ether) extract is used for extracting RNA of rice leaves of an experimental group and a control group respectively, and the RNA is subjected to reverse transcription cDNA to analyze expression quantity. The result shows that after the lotus plumule (petroleum ether) extracting solution is sprayed on rice inoculated with the bacterial blight disease for 14 days, the WRKY45 gene expression level is up-regulated by 1.8 times, the PR1b gene expression level is up-regulated by 2.25 times, and the PZ1 gene expression level is up-regulated by 3.67 times. FIG. 6 shows the detection of the expression level of the defense gene, and in FIG. 6, the detection of the expression levels of WRKY45, PZ1 and PR1b are shown from left to right.
In conclusion, in vitro bacteriostasis experiments and in vivo prevention and control experiments show that the lotus plumule extracting solution has strong bacteriostasis activity to the bacterial blight of the white leaf blight, after the lotus plumule extracting solution is sprayed on the rice inoculated with the bacterial blight of the white leaf blight, the expression levels of WRKY45 gene, PR1b gene and PZ1 gene are all up-regulated to different degrees, the effective bacteriostasis component of the lotus plumule can induce or cause the expression of defense genes in plants, and the basic resistance of the rice is increased.
Claims (7)
1. A lotus plumule extracting solution is characterized by comprising the following steps:
step 1: drying the lotus plumule, and grinding into powder;
step 2: dissolving the powder in the step 1 by using ethanol for the first time, performing ultrasonic treatment and centrifugation, and taking supernate;
and step 3: drying the supernatant obtained in the step 2 under reduced pressure, and dissolving with distilled water;
and 4, step 4: and (4) extracting the solution obtained in the step (3) by using chloroform, drying under reduced pressure again, and then adding ethanol for secondary dissolution to obtain the lotus plumule extracting solution.
2. The lotus plumule extract liquid according to claim 1, wherein: in the step 2, the first dissolution with ethanol is to mix the powdery lotus plumule with 90% ethanol according to a material-liquid ratio of 1: 15 are dissolved.
3. The lotus plumule extract liquid according to claim 1, wherein: the ethanol added in the step 4 is ethanol with the concentration of 90 percent.
4. The lotus plumule extract liquid according to claim 1, wherein: in step 4, n-butanol or petroleum ether is used to replace chloroform.
5. The lotus plumule extract as claimed in any one of claims 1 to 4, which has an inhibitory effect on rice bacterial blight.
6. The induction effect of the lotus plumule extract as claimed in any one of claims 1 to 4 on rice defense gene expression.
7. The lotus plumule extract liquid according to claim 6, wherein: the rice defense genes are WRKY45 gene, PR1b gene and PZ1 gene.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1257069A (en) * | 1998-12-14 | 2000-06-21 | 同济医科大学 | Process for extracting lotucine from plumula nelumbinis |
| CN103610756A (en) * | 2013-12-17 | 2014-03-05 | 重庆百农网投资有限公司 | Extraction method for total alkaloids of lotus plumule |
| CN104306455A (en) * | 2014-11-07 | 2015-01-28 | 中南民族大学 | Lotus plumule chloroform extract and preparation method and use thereof |
| CN107951928A (en) * | 2017-12-20 | 2018-04-24 | 广东工业大学 | The new opplication of Lotus Plumule P.E. |
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- 2019-11-15 CN CN201911120874.XA patent/CN110786341B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1257069A (en) * | 1998-12-14 | 2000-06-21 | 同济医科大学 | Process for extracting lotucine from plumula nelumbinis |
| CN103610756A (en) * | 2013-12-17 | 2014-03-05 | 重庆百农网投资有限公司 | Extraction method for total alkaloids of lotus plumule |
| CN104306455A (en) * | 2014-11-07 | 2015-01-28 | 中南民族大学 | Lotus plumule chloroform extract and preparation method and use thereof |
| CN107951928A (en) * | 2017-12-20 | 2018-04-24 | 广东工业大学 | The new opplication of Lotus Plumule P.E. |
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