CN109463386B - Application of mangostin in controlling bacterial wilt - Google Patents
Application of mangostin in controlling bacterial wilt Download PDFInfo
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- CN109463386B CN109463386B CN201811252610.5A CN201811252610A CN109463386B CN 109463386 B CN109463386 B CN 109463386B CN 201811252610 A CN201811252610 A CN 201811252610A CN 109463386 B CN109463386 B CN 109463386B
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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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
Abstract
The invention discloses an application of mangostin in controlling bacterial wilt. The invention finds that the gamma-mangostin and the alpha-mangostin have good inhibition effect on Ralstonia solanacearum, and after the gamma-mangostin or the alpha-mangostin is used for treating the Ralstonia solanacearum, the formation amount of a biological membrane of the Ralstonia solanacearum can be reduced, the cell length is obviously increased, the cell is deformed, the cell membrane is shrunk to different degrees, the cell morphology is damaged, and the growth and the propagation of the Ralstonia solanacearum are inhibited, so the gamma-mangostin and the alpha-mangostin can be used as an inhibitor or a pesticide preparation for resisting the Ralstonia solanacearum and the like to prevent and treat agricultural related diseases such as the Ralstonia solanacearum and the like, or can be used as a soil additive to.
Description
Technical Field
The invention belongs to the technical field of biological pesticides, and particularly relates to application of mangostin in bacterial wilt prevention and control.
Background
Ralstonia solanacearum (Ralstonia solanacearum) is a soil-borne pathogenic bacterium that can cause bacterial wilt in plants. The ralstonia solanacearum has extremely strong infectivity, global geographical distribution and an abnormally wide host range, and becomes one of the most destructive plant bacterial diseases, once the ralstonia solanacearum occurs, plant stems and leaves can wither and droop until all the plants die, so far, no effective control method exists, and the disease is called as the 'cancer' of the plants. Ralstonia solanacearum can be harmful to various plants, and is reported to infect more than 200 plants of 53 families in recent years. Ralstonia solanacearum is a complex strain with high variability and adaptability, and the physiological and biochemical pathogenic pathways are numerous, the complexity causes the bacterial wilt of plants to be difficult to prevent and treat, and the production and development of agriculture and forestry in tropical, subtropical and partial temperate regions are seriously threatened.
For a long time, the control measures of the ralstonia solanacearum mainly depend on chemical pesticides, resistant varieties, aseptic grafting, crop rotation and other measures, but the control effect is not ideal, and the problems of environmental pollution, resistance and the like exist. Therefore, the search for an efficient and green method for controlling bacterial wilt is always the focus of the agricultural research field. The natural product of plant source has the characteristics of structural diversity, multiple target points of action and difficult generation of drug resistance, and is always a research hotspot and trend in the field of pesticides at present. At present, a great deal of research is carried out on the chemical components of mangosteen at home and abroad, the mangosteen is rich in mangostin compounds, has the functions of resisting inflammation, oxidation, thrombus and tumor, and has the antibacterial function on staphylococcus aureus, escherichia coli, vibrio cholerae and other bacteria. The application research of mangosteen in agriculture is not much, the mangosteen is one of 'queen' in fruits, fresh food is taken as the main food, and a large amount of shells generated after the mangosteen is used are treated as garbage. However, until now, there has been no report on the use of mangostin for controlling diseases associated with bacterial wilt.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the application of mangostin in the control of bacterial wilt.
The invention also aims to provide the application of mangostin in preparing the ralstonia solanacearum inhibitor.
The purpose of the invention is realized by the following technical scheme: application of mangostin in preventing and treating bacterial wilt.
The mangostin is at least one of gamma-mangostin (gamma-mangostin) and alpha-mangostin (alpha-mangostin); preferably gamma-mangostin (gamma-mangosteen); the structural formula of the gamma-mangostin is shown as a formula I, and the structural formula of the alpha-mangostin is shown as a formula II:
the bacterial wilt prevention and control method is to kill bacterial wilt causing bacterial wilt or inhibit bacterial wilt causing bacterial wilt.
Application of mangostin in preparing ralstonia solanacearum inhibitor or pesticide preparation.
The mangostin is at least one of gamma-mangostin and alpha-mangostin; preferably gamma-mangostin.
The effective concentration of the gamma-mangostin in the ralstonia solanacearum inhibitor or the pesticide preparation is 8-500 mug/mL; preferably 16-500 mu g/mL; more preferably 16-128 mug/mL; most preferably 128. mu.g/mL.
The effective concentration of the alpha-mangostin in the ralstonia solanacearum inhibitor or the pesticide preparation is 16-500 mu g/mL; preferably 398.14-500 μ g/mL.
The mangostin is added into the soil to kill the ralstonia solanacearum in the soil or inhibit the ralstonia solanacearum in the soil.
The mangostin is at least one of gamma-mangostin and alpha-mangostin; preferably gamma-mangostin.
The mangostin is used as a soil additive in the preparation of biological preparations for controlling bacterial wilt.
The mangostin is at least one of gamma-mangostin and alpha-mangostin; preferably gamma-mangostin.
The biological agent comprises a microbial agent and the like.
Compared with the prior art, the invention has the following advantages and effects: according to the invention, waste mangosteen peels are fully utilized, the gamma-mangostin and the alpha-mangostin in the mangosteen are found to have good inhibition effect on Ralstonia solanacearum, and the gamma-mangostin and the alpha-mangostin are used as novel anti-ralstonia solanacearum inhibitors and can be used for preventing and treating agricultural related diseases such as bacterial wilt and the like.
Drawings
FIG. 1 is a graph showing the effect of various concentrations of gamma-mangosteen treatment on the growth curve of Ralstonia solanacearum.
FIG. 2 is a graph showing the effect of various concentrations of α -mangosteen treatment on the growth curve of Ralstonia solanacearum.
FIG. 3 is a graph showing the inhibition of Ralstonia solanacearum by gamma-mangosteen and alpha-mangosteen at different concentrations.
FIG. 4 is a graph of the effect of different concentrations of gamma-mangosteen treatment on Ralstonia solanacearum biofilms (in the graph: Duncan multiplex assay with the same letters indicated no significant difference at the P0.05 level, indicated as a, b, c, respectively).
FIG. 5 is a graph of the change in the bacterial biofilm of Ralstonia solanacearum at different treatment times for the same drug (. alpha. -mangosteen) (in the graph: the Duncan multiplex assay with the same letters indicates no significant difference at the P0.05 level, indicated as a, b, c, respectively).
FIG. 6 is a graph showing morphological changes of Ralstonia solanacearum cells under the influence of plant source compounds observed under a scanning electron microscope after gamma-mangosteen treatment of Ralstonia solanacearum; wherein, the graph a shows the bacterial status of a control group of Lawsonia inermis of Solanaceae without being treated by gamma-mangosteen; FIGS. b, c and d are bacterial status of Ralstonia solanacearum after gamma-mangosteen low concentration treatment (16. mu.g/mL); graphs e and f are the bacterial status after the gamma-mangosteen concentration reaches 128. mu.g/mL.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The gamma-mangostin (gamma-mangosteen) and the alpha-mangostin (alpha-mangosteen) related in the embodiment of the invention can be directly obtained from the market; can be obtained by chemical synthesis; or separating and extracting mangosteen pericarp by existing method.
The ralstonia solanacearum referred to in the examples of the present invention can be obtained commercially by a conventional method, or isolated from a plant infected with ralstonia solanacearum by a conventional method.
Example 1
1. Effect of gamma-mangosteen and alpha-mangosteen on the growth curves of Ralstonia solanacearum
(1) Preparing gamma-mangosteen (gamma-mangostin) and alpha-mangosteen (alpha-mangostin) into mother liquor of 200 mu g/mL respectively, and filtering for standby (diameter is 13mm, and pore diameter is 0.22 um);
(2) preparing NB culture solution (namely NB culture medium), and autoclaving for standby;
(3) the gamma-mangosteen and alpha-mangosteen stock solutions were pipetted into 30mL NB medium in a sterile operating station to give final concentrations of 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, respectively, and three replicates were set with a final concentration of 0.5% (v/v) DMSO (dimethylsulfoxide) solvent Control (CK);
(4) inoculating 100uL (OD value is 1.0) of bacterial liquid of Ralstonia solanacearum to 30mL of culture solution with medicine, and shake-culturing at 30 ℃ of a shaking table and 180 r/min;
(5) sampling every 3h in the culture process, and measuring and recording the absorbance of the sample at 600nm of an ultraviolet spectrophotometer until 24h after inoculation;
(6) the growth curve of ralstonia solanacearum under the influence of gamma-mangosteen and alpha-mangosteen is plotted with the sampling time as abscissa and the sample OD600 absorbance as ordinate, and statistical analysis is performed on SPSS17.0 (in brilliant plum, 2015).
The influence of the plant source compounds gamma-mangosteen and alpha-mangosteen on the growth of the bacterial cells of ralstonia solanacearum is measured, and the growth curve of ralstonia solanacearum in the presence of gamma-mangosteen and alpha-mangosteen is drawn. The results are shown in FIGS. 1 and 2:
observing the growth curve of the blank-processed ralstonia solanacearum, wherein the growth and reproduction period of the ralstonia solanacearum is 0-6 h under the culture condition, which is a delay period, and the bacteria grow less; entering a logistic growth phase of logistic for 6-12 hours, and enabling bacteria to grow in large quantity; after 12 hours the strain grew steadily and the rate of propagation slowed down gradually. Under experimental conditions, the plant source compounds gamma-mangosteen and alpha-mangosteen have different degrees of influence on the growth of the ralstonia solanacearum after being treated at different concentrations.
The effect on the growth curve of ralstonia solanacearum was shown with increasing concentration of the gamma-mangosteen treatment. The effect on the growth curve of the ralstonia solanacearum is not obviously shown at low concentration, and the delay of the logarithmic growth phase of the growth curve of the ralstonia solanacearum is shown at the concentrations of 100 mu g/mL and 200 mu g/mL, and the delay is 9-18 h from 6-12 h of a control group (figure 1). The ralstonia solanacearum growth curve under alpha-mangosteen treatment did not show significant differences from the blank control (fig. 2).
2. Bacteriostatic effect
(1) Thawed SMSA Medium (peptone 10g, glucose 10g, enzymatically hydrolyzed Casein 1g, ddH)2O1000mL, adjusting PH to 7.0, sterilizing at 121 ℃ for 20min), standing to about 35 ℃, adding ralstonia solanacearum liquid to make the final concentration of ralstonia solanacearum be OD600 ═ 0.1, fully mixing, pouring into culture dishes with 15mL each, and preparing into a bacterium-carrying plate;
(2) after the medium in the plate solidified, the plate was placed in an Oxford cup (inner diameter 6mm, outer diameter 8mm, height 10mm), and 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 80. mu.g/mL, 160. mu.g/mL of γ -mangosteen, α -mangosteen solution, 150. mu.L, with DMSO as a blank, were added thereto. Culturing the culture dish in a constant temperature incubator at 30 ℃ and 70% humidity for 48 h;
(3) and measuring the diameter of the inhibition zone after 48 hours, wherein the calculation mode of the inhibition rate is as follows:
(4) under the same culture conditions, the inhibition of ralstonia solanacearum by gamma-mangosteen and alpha-mangosteen at different concentrations (0, 8, 16, 32, 64, 128 mu g/mL) is greatly different. As a control, streptomycin sulfate was used at various concentrations (0, 8, 16, 32, 64, 128. mu.g/mL), and some results are shown in FIG. 3: under the condition of low concentration of 8 mu g/mL, the gamma-mangosteen shows a certain bacteriostatic effect, the bacteriostatic rate reaches 37.5 percent, the bacteriostatic rate is increased along with the increase of the concentration, and the bacteriostatic rate is up to 76.87 percent when the concentration is 128 mu g/mL. The alpha-mangosteen does not show obvious bacteriostatic effect under the condition of low concentration of 8 mu g/mL, and the bacteriostatic rate is only 18.75 percent at 128 mu g/mL.
3. Microplate assay for biofilm formation
(1) Taking 3 1.5mL sterile centrifuge tubes, wherein the labels are 1, 2 and 3, adding 793.8 mu L of NB culture solution into all the centrifuge tubes, then adding 42 mu L of DMSO into the tube 1, respectively adding 42 mu L of gamma-mangosteen mother solution with the concentration of 0.2mg/mL and 10mg/mL into the tubes 2 and 3, finally adding 4.2 mu L of ralston solution (OD600 is 1.0) into all the centrifuge tubes, uniformly mixing, adding 200 mu L of the mixed culture solution into each well of a 96-well plate (no culture is added into the edge well of the culture plate for eliminating the edge effect), repeating each well once, and repeating each treatment for six times;
(2) standing and culturing at 30 deg.C for 48 hr;
(3) carefully sucking the culture, adding sterile distilled water, gently washing for 2 times, and then discarding the distilled water;
(4) adding 220 μ L of crystal violet staining solution with concentration of 0.1% to stain the biological membrane, standing and staining for 30min at room temperature;
(5) after dyeing, removing crystal violet, washing twice with 200 mu L distilled water, removing floating color, and drying at room temperature for 30 min;
(6) 200. mu.L of 95% (v/v) ethanol was added to dissolve the crystal violet adsorbed on the biofilm for 30min, and the absorbance at 490nm (in Brilliant plum, 2015) was measured using a microplate reader.
Experiment the effect of two plant source compounds, gamma-mangosteen and alpha-mangosteen, on the biofilm forming ability of lawsonia solanacearum was investigated by measuring biofilm formation with a microplate reader at OD490 nm. The results show that under the experimental conditions, the biofilm formation amount of the ralstonia solanacearum after 24h and 48h of culture is not obviously different. OD490nm after 24h of culture was 0.246, biofilm formation after 48h was slightly but not significant, OD490nm was 0.270 and increased only by 0.024. The formation of the ralstonia solanacearum biofilm reaches a stable state after 24 hours, and a large increase cannot occur later.
The different effects of the two plant-derived compounds (gamma-mangosteen and alpha-mangosteen) on the formation of a cyanobacterium biofilm were studied at different concentrations or for different times:
the effect of gamma-mangosteen treatment at different concentrations on the cyanobacterial biofilm is shown in FIG. 4: after the gamma-mangosteen treats the ralstonia solanacearum for 24 hours, the biofilm formation amount of the strain is remarkably reduced compared with that of a control group under the low concentration of 10 mu g/mL; the reduction of the biofilm formation amount of the strain at a high concentration of 500. mu.g/mL was more significant than that of the control group and the low concentration-treated group. After 48h, the biofilm formation amount of the strain under the low-concentration treatment is remarkably reduced compared with that of the control group, and the biofilm formation amount of the strain under the high-concentration treatment is still remarkably reduced compared with that of the control group and the low-concentration treatment group.
② the change of the biological membrane of the ralstonia solanacearum under different treatment time by the same drug (alpha-mangosteen) is shown in figure 5: under the condition that 10 mu g/mL of alpha-mangosteen is used for treating ralstonia solanacearum, the biofilm formation amount of the strain 24h is remarkably smaller than that of the strain 48h, the OD490nm values are respectively 0.08 and 0.19, and after the alpha-mangosteen is used for treating the ralstonia solanacearum, the biofilm formation amounts of the strains at the same concentration and different time periods show remarkable difference.
4. Scanning electron microscope for observing morphological change of ralstonia solanacearum cells
(1) Adding 100 mu L of ralstonia solanacearum solution with OD600 of 1.0 into 30mL of NB culture solution with the medicine to ensure that the final concentration of the medicine (gamma-mangosteen) contained in the solution is two gradients of 128 mu g/mL and 16 mu g/mL, and performing shake culture for 24h at 30 ℃ in a shaking table at 180 r/min;
(2) 1.0mL of the drug-loaded ralstonia solanacearum solution with OD600 of 1.6 was aspirated and centrifuged in a high-speed centrifuge (Eppendorf AG22331 Hamburg) at 5000r/min for 8 min. Discarding the supernatant, adding 0.1M PBS buffer solution for rinsing, centrifuging, discarding the supernatant, and repeating for three times;
(3) sucking 500 mu L of 2.5% (v/v) glutaraldehyde by a pipette, blowing and beating the precipitate until the bacteria liquid is uniform, sucking a small amount of bacteria liquid to a cover glass, quickly and lightly coating the bacteria liquid uniformly, and putting the bacteria liquid into a refrigerator at 4 ℃ for fixing for 12 hours;
(4) rinsing with 0.1M PBS buffer for 3 times, each for 10 min;
(5) 1% (w/v) osmic acid was fixed in a fume hood for 30 min;
(6) rinsing with 0.1M PBS buffer for 3 times, each for 10 min;
(7) dehydrating with 30%, 50%, 70%, 80%, 90%, 100% (v/v) ethanol gradient, each gradient dehydrating for 10 min;
(8) drying in an oven overnight;
(9) and (5) observing the morphological change of the ralstonia solanacearum by a scanning electron microscope, and taking a picture for recording.
In the experiment, a ralstonia solanacearum sample treated by a plant source compound gamma-mangosteen is prepared by a cell biology method, the morphological change of the ralstonia solanacearum cell under the influence of the plant source compound is observed under a scanning electron microscope, the bacterial state of a control group of the ralstonia solanacearum which is not treated by the plant source compound is shown in figure 6a through observation under the scanning electron microscope, the bacterial cell is complete, the size is small, the size is close to that, the edge is obvious, and the surface is smooth. After gamma-mangosteen low concentration treatment (16. mu.g/mL) of Ralstonia solanacearum, as shown in FIGS. 6b, c and d, the cell length in the visual field is significantly increased, some cells are deformed, and the cell membrane is shrunk to different degrees, but the cell surface is still smooth. After the gamma-mangosteen concentration reaches 128 mug/mL, the bacterial state is as shown in FIGS. 6e and f, the cell surface is rugged, the cell membrane is rough and still has different degrees of shrinkage, the cell shape is seriously damaged and deformed, and the surface is accompanied by a plurality of bubble-shaped bulges, and the cell is further elongated.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. The application of mangostin in controlling bacterial wilt is characterized in that:
the mangostin is gamma-mangostin;
the effective concentration of the gamma-mangostin is 8-500 mu g/mL.
2. The use of mangostin according to claim 1 for controlling bacterial wilt, characterized in that:
the bacterial wilt prevention and control method is to kill bacterial wilt causing bacterial wilt or inhibit bacterial wilt causing bacterial wilt.
3. The application of mangostin in preparing the ralstonia solanacearum inhibitor is characterized in that:
the mangostin is gamma-mangostin;
the effective concentration of the gamma-mangostin in the ralstonia solanacearum inhibitor is 8-500 mu g/mL.
4. The use of mangostin according to claim 3 for the preparation of a ralstonia solanacearum inhibitor, characterized in that:
the effective concentration of the gamma-mangostin in the ralstonia solanacearum inhibitor is 16-128 mug/mL.
5. The application of mangostin as a soil additive in controlling bacterial wilt is characterized in that:
the mangostin is gamma-mangostin;
the effective concentration of the gamma-mangostin is 8-500 mu g/mL.
6. The application of mangostin as a soil additive in preparing a biological preparation for preventing and treating bacterial wilt is characterized in that:
the mangostin is gamma-mangostin;
the effective concentration of the gamma-mangostin is 8-500 mu g/mL.
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