CN109463386A - Application of the mangostin in prevention and treatment bacterial wilt - Google Patents
Application of the mangostin in prevention and treatment bacterial wilt Download PDFInfo
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- CN109463386A CN109463386A CN201811252610.5A CN201811252610A CN109463386A CN 109463386 A CN109463386 A CN 109463386A CN 201811252610 A CN201811252610 A CN 201811252610A CN 109463386 A CN109463386 A CN 109463386A
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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
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Abstract
The invention discloses application of the mangostin in prevention and treatment bacterial wilt.Find that γ-mangostin and α-mangostin have good inhibitory effect to Ralstonia solanacearum in the present invention, and after discovery γ-mangostin or α-mangostin processing Ralstonia solanacearum, the forming amount of Ralstonia solanacearum biomembrane can be reduced, cell length is caused to obviously increase, cellular dysmorphology, there is different degrees of shrinkage in cell membrane, cause cellular morphology impaired, and then inhibit the growth and breeding of Ralstonia solanacearum, therefore, it can be using γ-mangostin and α-mangostin as the inhibitor of anti-Ralstonia solanacearum or pesticidal preparations, for preventing and treating the related disease of the agricultural such as bacterial wilt, or as soil additive, it is added in soil, for preventing and treating bacterial wilt.
Description
Technical field
The invention belongs to biological pesticide technical field, in particular to application of the mangostin in prevention and treatment bacterial wilt.
Background technique
Ralstonia solanacearum (Ralstonia solanacearum) is a kind of native transmissibility that can cause plant-bacterial-wilt
Pathogenetic bacteria.Since Ralstonia solanacearum has extremely strong infectivity, global geographical distribution and abnormal extensive host range,
One of destructive maximum bacterial diseases of plants is had become, once morbidity can cause plant stem-leaf wilting sagging until all
It is withered, there is no effectively preventing method, referred to as " cancer " of plant still so far.Ralstonia solanacearum can cause harm various plants, report in recent years
Road its can infect 53 200 various plants of section.Ralstonia solanacearum (Ralstonia solanacearum) is one multiple again
Miscellaneous strain, variability and adaptability with height, and also its Physiology and biochemistry Pathogenic pathway is numerous, and these complexity cause to plant
Object bacterial wilt difficulty is prevented refractory, has seriously threatened the torrid zone, the agriculture and forestry production and development of subtropical zone and part Temperate Region in China.
For a long time, the control measure of Ralstonia solanacearum depend on chemical pesticide, using resistant variety, sterile transfer
It connects with measures such as crop rotations, the problems such as but its control efficiency is undesirable, while there is also environmental pollution and resistances.Therefore, it finds
A kind of bacterial wilt control method efficiently, green is always the focus in agricultural research field.The natural products of plant origin has
Structure diversity, effect target point it is more, it is not easy to the characteristics of generating drug resistance, be always current research focus in the field of pesticide and
Trend.The chemical component of mangosteen is largely studied both at home and abroad at present, be rich in mangosteen chlorins compound, have it is anti-inflammatory,
It is anti-oxidant, antithrombotic, antitumor, and there are antibacterial functions to bacteriums such as staphylococcus aureus, Escherichia coli, comma bacillus.
It is not that very much, mangosteen is one of " queen " in fruit, based on fresh food, after use about the application study of mangosteen agriculturally
The a large amount of shell of generation as garbage disposal.But up to the present, there has been no mangostin is used in prevention and treatment bacterial wilt phase
Close the report in disease.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide mangostin in prevention and treatment bacterial wilt
In application.
Another object of the present invention is to provide mangostins to prepare the application in Ralstonia solanacearum inhibitor.
The purpose of the invention is achieved by the following technical solution: application of the mangostin in prevention and treatment bacterial wilt.
The mangostin is γ-mangostin (γ-mangosteen) and α-mangostin (α-mangosteen)
At least one of；Preferably γ-mangostin (γ-mangosteen)；The structural formula of the γ-mangostin such as Formulas I institute
Show, α-mangostin structural formula is as shown in Formula II:
The prevention and treatment bacterial wilt is to kill the Ralstonia solanacearum for causing the Ralstonia solanacearum of bacterial wilt or inhibition to cause bacterial wilt.
Mangostin is preparing the application in Ralstonia solanacearum inhibitor or pesticidal preparations.
The mangostin is at least one of γ-mangostin and α-mangostin；Preferably γ-mangosteen
Element.
γ-mangostin effective concentration is 8~500 μ g/mL in the Ralstonia solanacearum inhibitor or pesticidal preparations；It is preferred that
For 16~500 μ g/mL；More preferably 16~128 μ g/mL；Most preferably 128 μ g/mL.
α-mangostin effective concentration is 16~500 μ g/mL in the Ralstonia solanacearum inhibitor or pesticidal preparations；It is preferred that
For 398.14~500 μ g/mL.
Application of the mangostin as soil additive in prevention and treatment bacterial wilt, mangostin is added in soil, can
To kill the Ralstonia solanacearum for causing bacterial wilt in the Ralstonia solanacearum or inhibition soil of cause in soil bacterial wilt.
The mangostin is at least one of γ-mangostin and α-mangostin；Preferably γ-mangosteen
Element.
Application of the mangostin as soil additive in the biological agent of preparation prevention and treatment bacterial wilt.
The mangostin is at least one of γ-mangostin and α-mangostin；Preferably γ-mangosteen
Element.
The biological agent includes microbial bacterial agent etc..
The present invention has the following advantages and effects with respect to the prior art: the present invention makes full use of mangosteen to discard pericarp,
It was found that γ-mangostin and α-mangostin have good inhibitory effect to Ralstonia solanacearum in mangosteen, by γ-mangosteen
Element and inhibitor new application of the α-mangostin as anti-Ralstonia solanacearum can be used for preventing and treating the agriculture related diseases such as bacterial wilt
Evil.
Detailed description of the invention
Fig. 1 is influence diagram of the γ-mangosteen processing to Ralstonia solanacearum growth curve of various concentration.
Fig. 2 is influence diagram of the α-mangosteen processing to Ralstonia solanacearum growth curve of various concentration.
Fig. 3 is the inhibition situation map of γ-mangosteen and α-mangosteen to Ralstonia solanacearum of various concentration.
Fig. 4 is that various concentration γ-mangosteen is handled to the influence diagram of Ralstonia solanacearum biomembrane (in figure: containing same word
Matrix shows that Duncan multiple check difference in P=0.05 level is not significant, is indicated respectively with a, b, c).
Fig. 5 be identical drug (α-mangosteen) Ralstonia solanacearum biomembrane under the different disposal time variation diagram (in figure:
It indicates that Duncan multiple check difference in P=0.05 level is not significant containing same letter, is indicated respectively with a, b, c).
Fig. 6 is observation of plant source compound influence under a scanning electron microscope after γ-mangosteen processing Ralstonia solanacearum
Lower Ralstonia solanacearum cellular morphology variation diagram；Wherein, figure a is the control group Ralstonia solanacearum bacterium handled without γ-mangosteen
State；Scheme b, c and d be Ralstonia solanacearum γ-mangosteen low concentration processing after (16 μ g/mL) bacterium bacterium state；Scheme e and f be
γ-mangosteen concentration reaches the bacterium bacterium state after 128 μ g/mL.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Unless stated otherwise, the present invention uses reagent, method and apparatus is the art conventional reagents, method and apparatus.
γ-mangostin involved in the embodiment of the present invention (γ-mangosteen) and α-mangostin (α-
Mangosteen commercially available acquisition) can directly be passed through；It can be obtained by chemical synthesis；Also the prior art can also be used from mountain
Separation and Extraction obtains in bamboo pericarp.
Ralstonia solanacearum involved in the embodiment of the present invention can be obtained by conventional commercial, or green withered from infection using conventional method
It separates and obtains in the plant of germ.
Embodiment 1
1, influence of the γ-mangosteen and α-mangosteen to Ralstonia solanacearum growth curve
(1) γ-mangosteen (γ-mangostin) and α-mangosteen (α-mangostin) are each configured to
The mother liquor of 200 μ g/mL filters spare (diameter 13mm, aperture 0.22um)；
(2) NB culture solution (i.e. NB culture medium) is configured, high pressure sterilization is spare；
(3) γ-mangosteen and the spare mother liquor of α-mangosteen are drawn with liquid-transfering gun respectively in aseptic operating platform
It is added in 30mL NB culture solution, making the final concentration with medicine culture solution is respectively 10 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ
Three weights are arranged with DMSO (dimethyl sulfoxide) solvent control (CK) of final concentration of 0.5% (v/v) in g/mL, 200 μ g/mL
It is multiple；
(4) spare Ralstonia solanacearum (Ralstonia solanacearum) bacterium solution 100uL (value=1.0 OD) is taken to be inoculated in 30mL band medicine culture solution
In, at 30 DEG C of shaking table, shake culture under 180r/min；
(5) primary every 3h sampling in incubation, the absorbance of sample is measured simultaneously at ultraviolet specrophotometer 600nm
Record, until after connecing bacterium for 24 hours；
(6) using sample time as abscissa, sample OD600 light absorption value is that ordinate is plotted in γ-mangosteen and α-
The growth curve chart of Ralstonia solanacearum under the influence of mangosteen, SPSS17.0 (Yu Yanmei, 2015) for statistical analysis.
Influence of the phytochemical γ-mangosteen and α-mangosteen to Ralstonia solanacearum thalli growth is measured, is drawn
Growth curve of the Ralstonia solanacearum processed in the case where having γ-mangosteen and α-mangosteen.As a result as depicted in figs. 1 and 2:
Observe Ralstonia solanacearum known to the growth curve of space management Ralstonia solanacearum under culture conditions, the period of strain growth breeding
It is lag phase for 0~6h, bacterial growth is less；Enter logistic logarithmic growth phase, bacterium raised growth within 6~12 hours；12
Bacterial strain stablizes growth after hour, and proliferative speed slows down gradually.Under experimental conditions, phytochemical γ-mangosteen
There is different degrees of influence to the growth of Ralstonia solanacearum with after the processing of α-mangosteen various concentration.
With the increase of γ-mangosteen concentration for the treatment of, the influence to Ralstonia solanacearum growth curve is shown.Under low concentration
And it is not apparent from the influence shown to Ralstonia solanacearum growth curve, and shown under the concentration of 100 μ g/mL and 200 μ g/mL green withered
The delay of bacterium growth curve logarithmic growth phase is 9~18h (Fig. 1) by 6~12h delay of control group.α-mangosteen processing
Under Ralstonia solanacearum growth curve be not apparent from and show and the difference of blank control group (Fig. 2).
2、IC50The measurement of (half Mlc)
(1) γ-mangosteen and α-mangosteen is big with inhibition zone to the measurement of the 503nhibiting concentration of Ralstonia solanacearum
It is small for according to and calculate；
(2) SMSA culture medium (peptone 10g, glucose 10g, enzyme hydrolysis the casein 1g, ddH melted2O1000mL,
PH 7.0 is adjusted, sterilize 20min at 121 DEG C) it places to 35 DEG C or so, Ralstonia solanacearum liquid is added and makes Ralstonia solanacearum final concentration of
OD600=0.1 is poured into culture dish after mixing well, and the plate that carries disease germs is made in every ware 15mL；
(3) it is put into Oxford cup (internal diameter 6mm, outer diameter 8mm, high 10mm) after culture medium solidification in plate, is separately added into 5 μ g/
ML, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL, 80 μ g/mL, the γ-mangosteen of 160 μ g/mL, α-mangosteen solution
150uL, using DMSO as blank control.Culture dish cultivates 48h 30 DEG C in constant incubator, under 70% humidity；
(4) antibacterial circle diameter is measured after 48h, the calculation of bacteriostasis rate is as follows:
Then it is statisticallyd analyze with SPSS17.0 and calculates IC50(Li et al.,2014)。
(5) under same culture conditions, the γ-mangosteen of various concentration (0,8,16,32,64,128 μ g/mL) and
α-mangosteen has larger difference to the inhibition situation of Ralstonia solanacearum.With various concentration (0,8,16,32,64,128 μ g/mL)
Streptomycin sulphate is control, and partial results are as shown in Figure 3: under conditions of 8 μ g/mL of low concentration, γ-mangosteen is shown
Certain fungistatic effect, bacteriostasis rate reach 37.5%, and with the increase of concentration, bacteriostasis rate is consequently increased, be in concentration
When 128 μ g/mL, bacteriostasis rate is up to 76.87%.α-mangosteen is not shown obviously under conditions of 8 μ g/mL of low concentration
Fungistatic effect, in 128 μ g/mL, bacteriostasis rate is only 18.75%.γ-mangosteen and α-mangosteen are to Ralstonia solanacearum
IC50Value is respectively 21.26 μ g/mL and 398.14 μ g/mL, and the results are shown in Table 1.
1 γ-mangosteen of table, α-mangosteen IC50The measurement of value
| Sample | Regression equation | IC50(μg/mL) | R value |
| γ–mangosteen | Y=0.278x+44.089 | 21.26 | 0.8965 |
| α-mangosteen | Y=0.104x+8.5938 | 398.14 | 0.6210 |
3, microwell plate measures biofilm formation
(1) 3 1.5mL sterile centrifugation tubes are taken, 793.8 μ L NB culture solutions are added in label 1,2,3 in all centrifuge tubes,
42 μ L DMSO are added in No. 1 pipe again, the γ-of 42 μ L 0.2mg/mL, 10mg/mL is separately added into 2, No. 3 pipes
4.2 μ L Ralstonia solanacearum liquid (OD600=1.0) are finally added, in 96 orifice plates after mixing in mangosteen mother liquor in all centrifuge tubes
In every hole the 200 above-mentioned mixed-culture mediums of μ L (in order to eliminate edge effect, culture is not added in the edge hole of culture plate) is added, each
Hole once repeats, six repetitions of each processing；
(2) stationary culture 48 hours at 30 DEG C；
(3) culture is carefully drawn, is added after sterile distilled water gently washs 2 times and discards distilled water；
(4) it is that 0.1% violet staining liquid dyes biomembrane that 220 μ L concentration, which are added, is stored at room temperature dyeing 30min；
(5) it after dyeing, removes crystal violet and is cleaned twice with 200 μ L distilled water, remove drying at room temperature 30min after loose colour；
(6) ethyl alcohol that 200 μ L 95% (v/v) are added dissolves the crystal violet 30min being adsorbed on biomembrane, utilizes enzyme mark
Instrument measures its light absorption value (Yu Yanmei, 2015) at 490nm.
Experiment measures the formational situation of biomembrane by microplate reader at OD490nm, studies γ-mangosteen and α-
Influence of the two kinds of phytochemicals of mangosteen to Ralstonia solanacearum biofilm formation ability.The results show that testing
Under the conditions of Ralstonia solanacearum culture have no significant difference with the biofilm formation amount after 48h for 24 hours.Culture for 24 hours after OD490nm be
Biofilm formation amount after 0.246,48h has increased slightly, but not significant, and OD490nm 0.270 only increases by 0.024.Show
The formation of Ralstonia solanacearum biomembrane has reached stable state after 24h, is later not in biggish growth.
It studies two kinds of phytochemicals (γ-mangosteen and α-mangosteen) various concentration or different time is made
There is different influences with the lower formation to Ralstonia solanacearum biomembrane:
1. influence result of the various concentration γ-mangosteen processing to Ralstonia solanacearum biomembrane is as shown in Figure 4: γ-
After mangosteen handles Ralstonia solanacearum for 24 hours, the biofilm formation amount of bacterial strain subtracts compared with control group conspicuousness at 10 μ g/mL of low concentration
It is few；The reduction of bacterial strain biofilm formation amount is in conspicuousness compared with control group and low concentration processing group under the high concentration of 500 μ g/mL.
After 48h, the bacterial strain biofilm formation amount under low concentration processing is reduced compared with control group conspicuousness, bacterial strain biofilm formation under high concentration
Amount is still in that conspicuousness is reduced compared with control group and low concentration processing group.
2. identical drug (α-mangosteen) variation of Ralstonia solanacearum biomembrane under the different disposal time is as shown in Figure 5:
10 μ g/mL α-mangosteen are handled under Ralstonia solanacearum, bacterial strain for 24 hours biofilm formation amount significantly less than 48h biofilm formation amount,
OD490nm value is respectively the bacterial strain life under same concentrations different time after 0.08,0.19, α-mangosteen processing Ralstonia solanacearum
Object film forming amount is to show significant difference.
4. scanning electron microscopic observation Ralstonia solanacearum cellular morphology changes
(1) make its contained drug (γ-with the 100 μ L of Ralstonia solanacearum liquid that OD600=1.0 is added in medicine NB culture solution in 30mL
Mangosteen) final concentration is two gradients of 128 μ g/mL and 16 μ g/mL, and training is shaken at 180r/min in shaking table, 30 DEG C
It supports for 24 hours；
(2) the band medicine Ralstonia solanacearum liquid of 1.0mL OD600=1.6 is drawn respectively in supercentrifuge (Eppendorf AG
8min is centrifuged under 5000r/min in 22331Hamburg).Give up supernatant, the rinsing of 0.1M PBS buffer solution is added, is abandoned after centrifugation
Supernatant is removed, in triplicate；
(3) pipette draws 500 μ L, 2.5% (v/v) glutaraldehyde and blows and beats that be precipitated to bacterium solution uniform, draws a small amount of bacterium solution
It drips on coverslip, quickly gently smoothens, be put into 4 DEG C of refrigerators and fix 12h；
(4) 0.1M PBS buffer solution rinses 3 times, each 10min；
(5) 1% (w/v) osmic acids fix 30min in draught cupboard；
(6) 0.1M PBS buffer solution rinses 3 times, each 10min；
(7) 30%, 50%, 70%, 80%, 90%, 100% (v/v) Gradient elution using ethanol, each serial dehydration 10min；
(8) it is put into oven dried overnight；
(9) it scanning electron microscopic observation Ralstonia solanacearum metamorphosis and photographs to record.
This experiment prepares phytochemical γ-mangosteen treated Ralstonia solanacearum using the means of cell biology
Sample, Ralstonia solanacearum cellular morphology changes under the influence of observation of plant source compound under a scanning electron microscope, tests by sweeping
Electric microscopic observation is retouched, the control group Ralstonia solanacearum bacterium state without phytochemical processing is as shown in Figure 6 a, bacterial cell
Completely, small volume and size are close, and edge is obvious, and surface is smooth.Ralstonia solanacearum is after the processing of γ-mangosteen low concentration
(16 μ g/mL) as shown in Fig. 6 b, c and d, cell length is obviously increased in the visual field, part cellular dysmorphology, and different journeys occurs in cell membrane
The shrinkage of degree, but cell surface is still smooth.After γ-mangosteen concentration reaches 128 μ g/mL, bacterium bacterium state such as Fig. 6 e
With shown in f, cell surface is uneven, and cell membrane is coarse and still with different degrees of shrinkage, cause cellular morphology seriously by
Damage, deformity, while surface, with many blister protrusions, cell further extends.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (9)
1. application of the mangostin in prevention and treatment bacterial wilt.
2. application of the mangostin according to claim 1 in prevention and treatment bacterial wilt, it is characterised in that:
The mangostin is at least one of γ-mangostin and α-mangostin.
3. application of the mangostin according to claim 1 in prevention and treatment bacterial wilt, it is characterised in that:
The prevention and treatment bacterial wilt is to kill the Ralstonia solanacearum for causing the Ralstonia solanacearum of bacterial wilt or inhibition to cause bacterial wilt.
4. mangostin is preparing the application in Ralstonia solanacearum inhibitor or pesticidal preparations.
5. mangostin according to claim 4 is preparing the application in Ralstonia solanacearum inhibitor or pesticidal preparations, feature
It is:
The mangostin is at least one of γ-mangostin and α-mangostin.
6. mangostin according to claim 5 is preparing the application in Ralstonia solanacearum inhibitor or pesticidal preparations, feature
It is:
γ-mangostin effective concentration is 8~500 μ g/mL in the Ralstonia solanacearum inhibitor or pesticidal preparations；
α-mangostin effective concentration is 16~500 μ g/mL in the Ralstonia solanacearum inhibitor or pesticidal preparations.
7. mangostin according to claim 6 is preparing the application in Ralstonia solanacearum inhibitor or pesticidal preparations, feature
It is:
γ-mangostin effective concentration is 16~128 μ g/mL in the Ralstonia solanacearum inhibitor or pesticidal preparations；
α-mangostin effective concentration is 398.14~500 μ g/mL in the Ralstonia solanacearum inhibitor or pesticidal preparations.
8. application of the mangostin as soil additive in prevention and treatment bacterial wilt, it is characterised in that: the mangostin is
At least one of γ-mangostin and α-mangostin.
9. application of the mangostin as soil additive in the biological agent of preparation prevention and treatment bacterial wilt, it is characterised in that: institute
The mangostin stated is at least one of γ-mangostin and α-mangostin.
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Cited By (1)
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| CN115886004A (en) * | 2022-12-19 | 2023-04-04 | 贵州大学 | Application of plant-derived natural product gamma-mangostin in antagonism of xanthomonas |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115886004A (en) * | 2022-12-19 | 2023-04-04 | 贵州大学 | Application of plant-derived natural product gamma-mangostin in antagonism of xanthomonas |
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