CN101270340A - Paris polyphylla var.yunnanensis endogenetic pichia for preparing volatile oil and antibiotic activity - Google Patents
Paris polyphylla var.yunnanensis endogenetic pichia for preparing volatile oil and antibiotic activity Download PDFInfo
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Abstract
The invention relates to a Paris polyphylla var. yunnanensis endogenetic Pichia pastoris used to produce volatile oil. The endogenetic fungi Ppf9 is acquired from Paris polyphylla var. yunnanensis by the endogenetic fungi isolation technique, and is classified and determined to be Pichia. The bacterial strain is preserved in China General Microbiological Culture Collection Center with the registered number of CGMCC No. 2475. Endogenetic Gliomastix used to produce volatile oil is isolated from Paris polyphylla var. yunnanensis for the first time herein, and the chemical compositions and the antibacterial activity of the volatile oil are identified. The invention is to make use of the endogenetic fungi resource of plant to acquire natural active ingredients.
Description
Technical field
The present invention relates to microbial technology field, relate to a kind of application of tool anti-microbial activity endogenetic fungus, give birth to pichia spp in microorganism-Rhizoma Paridis that particularly to relate to a kind of its meta-bolites be volatile oil.
Background technology
Plant endogenesis epiphyte (plant endophytic fungi) be meant those in its life history a certain stage or all stages live in health plant tissue or organ inside, host plant does not show the fungi of external disease symptom.Plant endogenesis epiphyte is a kind of new Microbial resources, can produce the meta-bolites of multiple structure type, has multiple biological activitys such as antibiotic, antiviral, antitumor, pest-resistant, coordinate plant growth.The meta-bolites that can produce novel structure, function uniqueness owing to plant endogenesis epiphyte becomes the potential resources of new compound, novel drugs, and they have important application prospects in fields such as agricultural, medicine, industry, food.Some plant endogenesis epiphytes of discovered in recent years can produce volatile composition (volatilecomponents), and these volatile components have antibiotic and insecticidal activity, can be applied to store in a warehouse and the prevention and control of plant diseases, pest control in fruits and vegetables plantation as fumigant.
Rhizoma Paridis (Paris polyphylla var.yunnanensis Hand.-Mazz.) belongs to unifacial leaf guiding principle (Monocotyledoneae) Liliales (Liliales) Trilliaceae (Trilliaceae) Paris in classification, it is the distinctive a kind of rare medicinal plant of China, cry Rhizoma Paridis, Herba Typhonii gigantei again, mainly being distributed in provinces and regions such as Yunnan, Sichuan, Guizhou, is the main raw material of Chinese patent medicines such as famous " Yunnan white powder ", " tranquilizing uterine blood ", " Ji Shengde antivenom tablet ", " the heat poison is clear ".Rhizoma Paridis is a kind of per nnial herb, its root stock perennation is underground, soil-borne pathogen had strong resistivity, this is relevant with its symbiotic endogenetic fungus probably, these endogenetic fungus can produce the invasion and attack that antibacterial substance is assisted Rhizoma Paridis opposing pathogen, promote its healthy growth.Have not yet to see the report that from the Rhizoma Paridis endogenetic fungus, extracts volatile component.The present invention focuses on the research of extraction, chemical composition analysis and the anti-microbial activity of endogenetic fungus volatile oil, for the physiological ecological function of inquiring into endogenetic fungus (as the disease resistance that improves the host etc.) and the exploitation of new type bactericide provide foundation.
The purpose of this invention is to provide and a kind ofly can produce the microorganism of volatile oil, promptly give birth to pichia spp in the Rhizoma Paridis through fermentation culture.
Summary of the invention
The present invention relates to a kind of pichia spp (Pichia sp.), the bacterial strain code name is Ppf9, now is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preservation date is on April 29th, 2008, and registering on the books is numbered 2475.Separation and the classification that the present invention relates to pichia spp identified, the preparation method and the compositional analysis of volatile oil, and volatile oil provides following technological method to the inhibition activity of pathogenic fungi and bacterium.
1, the separation of interior living pichia spp Ppf9
Gather fresh paris polyphylla rhizome, adopt the separation and the purification technique of endogenetic fungus, obtain a strain endogenetic fungus, be numbered Ppf9.
2, the morphological specificity of interior living pichia spp Ppf9
(1) asexual generation: endogenetic fungus Ppf9 bacterial strain is 25 ℃ of cultivations on the PDA substratum, bacterium colony densification, irregular projection, poor growth, there is fold at the back, and is unicellular, and pseudohypha is arranged, the cell oval, ellipse, colourless, big or small 2.2 μ m～4.8 μ m * 2 μ m～4.4 μ m.The morphological specificity of endogenetic fungus Ppf9 (seeing Fig. 1, Fig. 2 and Fig. 3) meets the morphological specificity of Pichia (Pichia) fungi.
(2) sexual generation: do not find.
3, the molecular biological characteristics of interior living pichia spp Ppf9
The utilization round pcr, amplification employing ITS universal primer ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 ') and ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ').The ITS sequence of Ppf9 bacterial strain is submitted to registration acquisition sequence number (accession number) EF495244 in the GenBank nucleotide sequence storehouse.Simultaneously the ITS sequence of other fungies among the ITS sequence of Ppf9 bacterial strain and the GenBank is compared, it is the highest to find out similarity, the fungi that sibship is nearest, the ITS sequence similarity degree of the ITS sequence of Ppf9 bacterial strain and Ji Shi pichia (Pichia guilliermondii) DQ663478 is 99%, and the confidence level of gathering in a branch is 100%.The phylogeny tree graph of its homology is seen Fig. 4.
4, the fermentation culture feature of interior living pichia spp Ppf9
Substratum: liquid potato-glucose (PD) substratum.
Culture temperature: 25 ± 1 ℃.
Whether illumination: not.
Incubation time: 7 days.
Shaking speed: 150rpm.
Cultural characteristic: cultivated the 3rd day, bacterium liquid is colourless, and thalline is gathered into spherical; Cultivated the 5th day, bacterium liquid is slightly orange red; Cultivated the 7th day, bacterium liquid is scarlet, and it is coccoid that mycelia becomes.
5, the preparation of interior living pichia spp Ppf9 volatile oil
Adopt in the hydrodistillation preparation and give birth to pichia spp volatile oil, yield is 0.17% (g/g fresh weight)
6, the chemical composition analysis of interior living pichia spp Ppf9 volatile oil
Adopt the method for gas chromatography-mass spectrum (GC-MS) coupling, from interior living pichia spp Ppf9 volatile oil, identify 27 compounds (table 1) altogether, account for 98.219% of total content, wherein the higher chemical ingredients of content is respectively: 1,1,3a, 7-Tetramethyl-1a, 2,3,3a, 4,5,6,7b-octahydro-1H-cyclopropa[a] naphthalene (25.896%), palmitinic acid (Palmiticacid, 15.514%), 1-Methyl-2,4-di (prop-1-en-2-yl)-1-vinylcyclohexane (7.914%), (E)-Octadec-9-enoic Acid ((E)-Octadec-9-enoic acid, 7.277%), (9E, 12E)-Linolenic Acid, the own ester of 12-diolefinic acid ((9E, 12E)-Ethyl octadeca-9,12-dienoate, 5.199%), 3,6a, 10-Trimethyl-2,3,4,5,6,6a, 7,8-octahydro-4,10a-methano-10aH-1-benzoxocin (4.813), 1-vinyl hexanol (1-Vinylhexanol, 4.212%), (-)-globulol ((-)-Globulol, 3.440%), (E)-Octadec-9-enoic Acid ethyl ester ((E)-Ethyloctadec-9-enoate, 3.032%).
7, the restraining effect to bacterial growth of interior living pichia spp Ppf9 volatile oil
Adopt porous plate-MTT development process, give birth to the inhibition activity of pichia spp Ppf9 volatile oil in measuring four kinds of gram negative bacteriums (intestinal bacteria, Agrobacterium tumefaciens, tomato bacterial spot germ, angular leaf spot of cucumber bacterium) and two kinds of gram positive bacteriums (subtilis, staphylococcus haemolyticus).Ppf9 volatile oil is to intestinal bacteria, Agrobacterium tumefaciens, tomato bacterial spot germ, the angular leaf spot of cucumber bacterium, subtilis, the minimum inhibitory concentration of staphylococcus haemolyticus (MIC) is respectively: 0.60mg/mL, 1.00mg/mL, 0.40mg/mL, 0.80mg/mL, 1.00mg/mL and 1.50mg/mL, and the positive control Vetstrep is to intestinal bacteria, Agrobacterium tumefaciens, tomato bacterial spot germ, the angular leaf spot of cucumber bacterium, subtilis, the minimum inhibitory concentration of staphylococcus haemolyticus (MIC) is respectively: 0.080mg/mL, 0.060mg/mL, 0.040mg/mL, 0.060mg/mL, 0.10mg/mL and 0.125mg/mL.
8, interior living pichia spp Ppf9 volatile oil is to the restraining effect of rice blast fungus spore germination
Interior 503nhibiting concentration (the IC that gives birth to pichia spp Ppf9 volatile oil to the rice blast fungus spore germination
50) be 1.558mg/mL, the positive control derosal is to the 503nhibiting concentration (IC to the rice blast fungus spore germination
50) be 0.00211mg/mL.
Description of drawings
Fig. 1: endogenetic fungus Ppf9 bacterium colony front.
Fig. 2: the endogenetic fungus Ppf9 bacterium colony back side.
Fig. 3: the cellular form of endogenetic fungus Ppf9.
Fig. 4: the phylogeny tree graph of endogenetic fungus Ppf9 homology.
Fig. 5: the gas chromatogram of endogenetic fungus Ppf9 volatile oil.
Fig. 6: normal alkane C
8～C
40Gas chromatogram.
Advantage of the present invention: the present invention is first clear and definite has a microbiology classification position of the interior living Pichia pastoris Ppf9 bacterial strain of broad spectrum antibiotic activity, Find that simultaneously this bacterium volatile oil is to Escherichia coli, Agrobacterium tumefaciens, tomato bacterial spot germ, avenae subsp.citrull, bacillus subtilis and molten The staphylococcic growth of blood has obvious inhibitory action, and spore germination has obvious inhibitory action to rice blast fungus. The objective of the invention is to utilize and plant Thing endogenetic fungus resource in the hope of obtaining the natural antibacterial active component, is the research and development of fungal source agricultural chemicals, for inquiring into the physiological ecological of endogenetic fungus Function provides foundation.
In order to understand better the present invention, further specify essentiality content of the present invention below in conjunction with embodiments of the invention, but content of the present invention Be not limited thereto.
Embodiment
Embodiment 1: the separation of endogenetic fungus Ppf9
Gather fresh paris polyphylla rhizome, the surface is earlier with 70% Ethanol Treatment 30s, handle 20min to carry out surface sterilization completely with 0.2% mercury chloride then, aseptic condition is removed epidermis down, with the piece section of root stock branch into about 0.5cm * 0.5cm * 0.5cm size, be placed on the PDA substratum, 1 in every ware, at 25 ℃, be cultured to the 7th～20 day under the illumination condition, carry out purifying from a bit of mycelium inoculation of edge picking of each bacterium colony to the PDA substratum, continuous purification 4～5 times is to the colonial morphology unanimity.Bacterial strain behind the purifying is preserved on the PDA inclined-plane, and wherein a strain endogenetic fungus is numbered Ppf9.
Embodiment 2: the morphological specificity of endogenetic fungus Ppf9 bacterial strain is observed
Endogenetic fungus Ppf9 bacterial strain is 25 ℃ of cultivations on the PDA substratum, the bacterium colony densification, and irregular projection, poor growth, there is fold at the back, and is unicellular, and pseudohypha is arranged, the cell oval, ellipse, colourless, big or small 2.2 μ m～4.8 μ m * 2 μ m～4.4 μ m.The morphological specificity of endogenetic fungus Ppf9 is seen Fig. 1～Fig. 3.Under culture condition, do not find the sexual generation of Ppf9.
Embodiment 3: ITS sequential analysis of endogenetic fungus Ppf9 bacterial strain and Molecular Identification
At first will transfer in the PDA liquid nutrient medium by the mycelia of activatory Ppf9 bacterial strain on flat board, suspension culture obtains fresh mycelia after 5 days, adopts conventional molecular biology method to extract genomic dna.The utilization round pcr, DNA cloning employing ITS universal primer ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 ') and ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ').The used primer of dna sequencing is respectively ITS1 and ITS4, adopts ABIPRISM 3730 sequenators, carries out forward (5 ' → 3 ') and oppositely (3 ' → 5 ') two-way order-checking respectively.
Reverse sequence forms 5 ' → 3 ' complete sequence with the forward sequence assembly behind DNAMAN software reverse complemental.The ITS rDNA sequence of gained is submitted to GenBank database (National Center for Biotechnology Information Website, http://www.ncbi.nlm.nih.gov), obtain sequence number (accession number) EF495244.Utilize the Blast instrument to carry out sequence alignment, find out sequence information with the highest bacterium of this sequence similarity degree.The ITS sequence similarity degree of the ITS sequence of Ppf9 bacterial strain and Ji Shi pichia (Pichia guilliermondii) DQ663478 is 99%, and the phylogeny tree graph of its homology is seen Fig. 4.
According to the Blast comparison result, therefrom extract with the sequence of the high bacterial strain of isolating endogenetic fungus Ppf9 bacterial strain sequence similarity, adopting ClustalX 1.8 softwares to carry out sequence aims at, use MEGA computed in software genetic distance again, utilize apart from the ortho position in the dependence method---------------------phase connection (Neighbor-joining then, NJ) make up evolutionary tree, calculate the Bootstrap value and come evaluation system to grow the reliability of tree.The phylogeny tree graph of Ppf9 homology is seen Fig. 4, and the confidence level that Ppf9 bacterial strain and Ji Shi pichia (Pichia guilliermondii) gather in a branch is 100%.
Embodiment 4: the zymotechnique of endogenetic fungus Ppf9 bacterial strain
What the present invention adopted is shaker fermentation, contain potato-glucose (PD) substratum 200mL in the triangular flask of 500mL volume, cultured Ppf9 mycelium inoculation is in sterilized substratum in advance, the dark cultivation 7 days under 25 ℃, 150rpm, get total fermented liquid 10L, adopt the method for high speed centrifugation (centrifugal force is 8000g, centrifugation time 10min) that mycelia and bacterium liquid are separated, get mycelium 150.0g, standby in-20 ℃ of preservations.
Embodiment 5: the preparation of endogenetic fungus Ppf9 volatile oil
Endogenetic fungus Ppf9 volatile oil adopts the hydrodistillation preparation.The zymotechnique of pressing among the embodiment 2 obtains fresh mycelia 150.0g, carries out the water distillation, and temperature is 100 ℃, collects volatile oil behind the continuous still battery 4h, keeps in Dark Place in 4 ℃.In profit blended effluent liquid (5.0mL), add a certain amount of sodium-chlor (NaCl) (10.0mg), extract with ether and (to repeat 3 times, each 10mL), use the dry back of anhydrous sodium sulphate (3.0g) concentrated extract (allowing ether volatilize naturally) then, the pure volatile oil that obtains is kept in Dark Place in 4 ℃ of sealings.
Calculate oil yield (g/g fresh weight):
Embodiment 6: the chemical composition analysis of endogenetic fungus Ppf9 volatile oil
The GC condition: chromatographic column is the HP-5 of Agilent company (Agilent) quartz capillary column [30m * 0.25mm * 0.25 μ m (5% phenyl)-methyl polysiloxane].250 ℃ of injector temperatures, 240 ℃ of transmission line temperature.Temperature programming is as follows: 50 ℃ of initial column temperatures, and stop 1.50min and rise to 180 ℃ with 10 ℃/min, stop 2min; Rise to 280 ℃ with 6 ℃/min then, stop 10min.Carrier gas is high-pure helium (99.999%); Post flow 1mL/min.Prepare endogenetic fungus Ppf9 volatile oil with embodiment 3 described methods, the volatile oil sample size is 1.0 μ L (splitting ratios: 1: 20) at every turn.The gas chromatogram of endogenetic fungus Ppf9 volatile oil is seen Fig. 5.
GC-MS analyzes: adopt Agilent 6890 GC-5973 MSD gas chromatograph-mass spectrometers to finish.The GC condition as mentioned above.The MS condition is as follows: 280 ℃ of ion source temperatures; Ionization mode EI, ionizing energy 70eV; Mass scanning scope m/z:50～500.
Retention factor (Retention indices, mensuration RI): serial normal alkane C
8-C
40(Sigma) with 50 times of chloroform dilutions, separate record C then with the same sample introduction of above-mentioned GC condition
8～C
40Each normal alkane retention time.Calculate the RI value of each composition with the linear temperature increase formula: RI=100n+100 (lgt
x-lgt
n)/(lgt
N+1-lgt
n), t wherein
x, t
nAnd t
N+1Be respectively analyzed component and carbonatoms and be in normal alkane (t between n and the n+1
n＜t
x＜t
N+1) eluting peak retention time (min).Normal alkane C
8～C
40Gas chromatogram see Fig. 6.
The retrieval of database: each component peaks relative content (Relative amounts, definite employing peak area normalization method RA).Each component peaks is retrieved with NIST Library (2002 editions) mass spectral database, compares with pertinent literature RI value simultaneously, and last is best qualification result with mass spectrum matching degree and the highest chemical structure of RI value matching degree; No RI value matching be qualification result with the highest chemical structure of mass spectrum matching degree.The GC-MS analytical results sees Table 1.27 compounds from endogenetic fungus Ppf9 volatile oil, have been identified altogether, account for 98.219% of total content, wherein the higher chemical ingredients of content is respectively: 1,1,3a, 7-Tetramethyl-1a, 2,3,3a, 4,5,6,7b-octahydro-1H-cyclopropa[a] naphthalene (25.896%), palmitinic acid (Palmitic acid, 15.514%), 1-Methyl-2,4-di (prop-1-en-2-yl)-1-vinylcyclohexane (7.914%), (E)-Octadec-9-enoic Acid ((E)-Octadec-9-enoic acid, 7.277%), (9E, 12E)-Linolenic Acid, own the ester ((9E of 12-diolefinic acid, 12E)-Ethyloctadeca-9,12-dienoate, 5.199%), 3,6a, 10-Trimethyl-2,3,4,5,6,6a, 7,8-octahydro-4,10a-methano-10aH-1-benzoxocin (4.813), 1-vinyl hexanol (1-Vinylhexanol, 4.212%), (-)-globulol ((-)-Globulol, 3.440%), (E)-Octadec-9-enoic Acid ethyl ester ((E)-Ethyl octadec-9-enoate, 3.032%).
The chemical ingredients GC-MS analytical results of table 1 endogenetic fungus Ppf9 volatile oil
Embodiment 7: the antibacterial activity of endogenetic fungus Ppf9 volatile oil
(1) bacterial isolates of determination of activity: subtilis (Bacillus subtilis ATCC 11562) (Gram-positive); Staphylococcus haemolyticus (Staphylococcus haemolyticus ATCC 29970) (Gram-positive); Angular leaf spot of cucumber bacterium (Pseudomonas lachrymansATCC 11921) (Gram-negative); Agrobacterium tumefaciens (Agrobacterium tumefaciens ATCC 11158) (Gram-negative); Intestinal bacteria (Escherichia coli ATCC 25922) (Gram-negative); Tomato bacterial spot germ (Xanthomonas vesicatoria ATCC11633) (Gram-negative).
(2) substratum: employing LB nutrient agar (yeast extract 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, pH 7.0) and the LB liquid nutrient medium (yeast extract 5g/L, peptone 10g/L, sodium-chlor 5g/L, pH 7.0).
(3) preparation of endogenetic fungus Ppf9 volatile oil solution: take by weighing Ppf9 volatile oil 40.0mg in the Eppendorf pipe, add the acetone (100%) of 0.3mL, add the 0.7mL sterilized water again, vibration is dissolved, and obtains the Ppf9 volatile oil mother liquor of 40.0mg/mL.The volatile oil mother liquor being mixed with series concentration then is 20.00mg/mL, 17.50mg/mL, 15.00mg/mL, 12.50mg/mL, 10.00mg/mL, 8.00mg/mL, 6.00mg/mL, 4.00mg/mL, 2.00mg/mL, 1.00mg/mL, 0.50mg/mL, 0.25mg/mL, and solvent is the solution of 30% acetone.Positive control is selected Vetstrep (Amresco) for use, takes by weighing Vetstrep 4.0mg in the Eppendorf pipe, and to wherein adding the 1.0mL sterilized water, vibration is dissolved, and obtains the Vetstrep mother liquor of 4.0mg/mL.Then the Vetstrep mother liquor is mixed with the solution that series concentration is 2.0mg/mL, 1.50mg/mL, 1.25mg/mL, 1.00mg/mL, 0.80mg/mL, 0.60mg/mL, 0.40mg/mL, 0.20mg/mL, 0.10mg/mL, 0.050mg/mL, 0.010mg/mL.
(4) anti-microbial activity is measured: antibacterial activity is measured employing porous plate-its minimum inhibitory concentration of MTT determination of color, and (Minimal inhibitoryconcentration MIC), the results are shown in Table 2.The concrete operations step is as follows: adding concentration in 96 well culture plates of cleaning sterile is 10
6The confession examination bacterium liquid 90 μ L of cfu/mL, the volatile oil 10 μ L of different concns gradient, the detection final concentration that obtains Ppf9 volatile oil like this is 2.0mg/mL, 1.75mg/mL, 1.50mg/mL, 1.25mg/mL, 1.00mg/mL, 0.80mg/mL, 0.60mg/mL, 0.4mg/mL, 0.20mg/mL, 0.10mg/mL, 0.05mg/mL, mg/mL 0.025mg/mL, and the solvent final concentration is the solution of 3.0% acetone.And the detection final concentration of positive control Vetstrep is 0.20,0.15mg/mL, 0.125mg/mL, 0.10mg/mL, 0.080mg/mL, 0.060mg/mL, 0.040mg/mL, 0.020mg/mL, 0.010mg/mL, 0.0050mg/mL, 0.0010mg/mL.Establish blank and solvent control (3.0% acetone) simultaneously, each handles 3 repetitions.96 orifice plates are added MTT (0.5mg/mL) solution 10 μ L in every hole after 37 ℃ are cultivated 24h down, after continuing to cultivate 4h, add 10%DMSO 100 μ L termination reactions, vibration 30min is to help its dissolving.Naked eyes can be observed the MIC value of judging Ppf9 volatile oil.For further checking MIC value, from each hole of porous plate, draw 10 μ L nutrient solutions and be applied on the LB flat board, observation statistics flat-plate bacterial colony number gets final product after cultivating 24h under 37 ℃.
The antibacterial activity of table 2 endogenetic fungus Ppf9 volatile oil
Embodiment 8: the restraining effect to the rice blast fungus spore germination of endogenetic fungus Ppf9 bacterial strain volatile oil
(1) substratum: employing oat tomato substratum (Oat-tomato agar medium, OTA).The step of oat tomato substratum of preparation 1L is: with full ripe tomato chopping, squeeze the juice with juice extractor, juice is with four layers of filtered through gauze, and it is stand-by to get filtering juice 150mL.Simultaneously, take by weighing rolled oats 30g, add the 1000mL deionized water, boil 30min, note stirring while boiling.The two-layer filtered through gauze of well-done rolled oats.Collect filtrate, add off-the-shelf tomato juice 150mL and 20g agar then in filtrate, heating and melting is used filtered through gauze while hot, adds deionized water then and is settled to 1000mL.121 ℃ of moist heat sterilization 20min after the packing.This substratum is used for the cultivation of rice blast fungus.
(2) for the plant pathogenic fungi of trying: rice blast fungus (Magnaporthe oryzae).
(3) rice blast fungus activation culture: the inoculating needle a small amount of mycelia of picking from the test tube of preserving bacterial classification with sterilization, be inoculated on the oat tomato juice culture medium flat plate, seal with sealing film, cultivate down for 25 ℃.
(4) rice blast fungus succeeding transfer culture: the tomato juice of falling oat culture medium flat plate, the about 40mL of every ware.Drip sterilized water 1mL with liquid-transfering gun in rice blast fungus bacterium colony central authorities, clean the bacterium colony surface gently with the inoculating needle after the sterilization then, note firmly wanting light, in order to avoid the galling media surface.After treating that sterilized water becomes muddiness, draw 100 μ L with liquid-transfering gun again and be added drop-wise to good oat tomato juice culture medium flat plate surface, be coated with rod with glass then and it be coated with evenly diffusing.After treating the media surface drying, build and be inverted 25 ℃ of cultivations down.
(5) the product spore is induced in mechanical stimulus: after cultivating 48h under the inoculation activatory rice blast fungus room temperature, drip the about 3mL of sterilized water on its surface, clean mycelia gently with the cotton swab after the sterilization then, mycelia machinery is wiped broken, clean continuously twice, treat to get final product after the grey bacterium colony becomes black, the water on surface is outwelled, and back-off dries moisture on the thieving paper after the sterilization then, with the gauze after three layers of sterilization the ware mouth is sealed, placed drying to have to cultivate 48h under the condition of light to obtain the rice blast fungus spore.
(6) preparation Ppf9 volatile oil solution: take by weighing Ppf9 volatile oil 20.0mg in the Eppendorf pipe, add the acetone (100%) of 0.3mL, add the 0.7mL sterilized water again, vibration is dissolved, and obtains the Ppf9 volatile oil mother liquor of 20.0mg/mL.Then Ppf9 volatile oil mother liquor being mixed with series concentration is 4.0mg/mL, 3.5mg/mL, 3.0mg/mL, 2.5mg/mL, 2.0mg/mL, 1.5mg/mL, 1.0mg/mL, 0.5mg/mL, 0.2mg/mL, 0.1mg/mL, 0.05mg/mL, 0.025mg/mL, and the solvent of dilution usefulness is 30% acetone soln.
Positive control adopts derosal (Aldrich), takes by weighing derosal 1.0mg in the Eppendorf pipe, and to the acetone soln 1.0mL that wherein adds 30%, vibration is dissolved, and obtains the derosal mother liquor of 1.0mg/mL.The derosal mother liquor being mixed with series concentration then is 0.03mg/mL, 0.02mg/mL, 0.016mg/mL, 0.010mg/mL, 0.004mg/mL, 0.002mg/mL, 0.001mg/mL, and solvent is 30% acetone soln.
(7) preparation spore suspension: produce dropping sterilized water 3.0mL on the good flat board of spore in mechanical induction, clean the bacterium colony surface gently with aseptic cotton carrier then, wash spore, the spore washing lotion is installed in the centrifuge tube of 1.0mL, centrifugal 3 times (centrifugal force 8000g, each centrifugal 10min).Prepare spore suspension then, utilize blood cell counting plate to measure spore concentration, spore concentration is formulated as 2 * 10
6Individual/mL.
(8) carry out determination of activity: prepare clean culture dish, in each culture dish, put four layers of thieving paper then, sterilize.After the sterilization, in every ware, add sterilized water 5.0mL, two aseptic toothpicks of parallel placement on thieving paper.Other is ready to depression slide, and the alcohol immersion 24h with 75% dries then.On each pothole, drip the rice blast fungus spore suspension (2 * 10 that 25 μ L prepare
6Individual/mL) the Ppf9 volatile oil solution with 25 μ L series concentration gradients (should fully shake on vibrator before dripping, to guarantee spore suspension and the abundant mixing of liquid medicine), the detection final concentration that obtains Ppf9 volatile oil like this is 2.0mg/mL, 1.75mg/mL, 1.5mg/mL, 1.25mg/mL, 1.0mg/mL, 0.75mg/mL, 0.5mg/mL, 0.25mg/mL, 0.10mg/mL, 0.05mg/mL, 0.025mg/mL, 0.0125mg/mL.And the detection final concentration of positive control is 0.015mg/mL, 0.01mg/mL, 0.008mg/mL, 0.005mg/mL, 0.002mg/mL, 0.001mg/mL, 0.0005mg/mL.Establish blank and solvent control (15% acetone) simultaneously, each handles 3 repetitions.Then depression slide is placed on the toothpick in the culture dish, observes behind the cultivation 7h under the room temperature.
(9) calculating of observed and recorded experimental result and spore germination rate
Spore germination situation on doubly following each depression slide of observation of microscope 10 * 10 (eyepiece * object lens), select 3 different visuals field, 100 above spores in each visual field, be total to several 300 spores, write down the spore count of sprouting with counter, repeat to be incorporated into one with 3 and calculate spore germination rate (%).
(10) calculating of data processing and 503nhibiting concentration
Test-results adopts Microsoft Excel software to carry out data preparation, in analysis, to take the logarithm (X) for test agent concentration, inhibiting rate is converted into biometrics probability value (Y), try to achieve virulence regression equation (Y=aX+b), thus can be in the hope of 503nhibiting concentration (IC
50).Blank and solvent control (15% acetone) are established in experiment, and each handles two repetitions.
Ppf9 volatile oil is to the equation of linear regression of rice blast fungus spore germination: Y=1.8262X+4.6482 (coefficient R=0.9735); IC
50=1.558mg/mL.
The positive control derosal is to the equation of linear regression of rice blast fungus spore germination: Y=1.6113X+9.3123 (coefficient R=0.9813); IC
50=0.00211mg/mL.
Claims (6)
1, the Rhizoma Paridis endogenetic fungus of volatile oil is produced in a strain, it is characterized in that called after pichia spp (Pichia sp.), has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and registering on the books is numbered CGMCC No.2475.
2, a kind of separation method by the described endogenetic fungus of claim 1 by the surface sterilization of strictness, is separated from inside plants, and by form and Molecular Identification, ITS rDNA sequence is submitted to the GenBank database, obtains sequence number EF495244.
3, a kind of Accessory Right requires the volatile oil that obtains in the 1 described endogenetic fungus, and this volatile oil has anti-microbial activity.
4, a kind of method for preparing the endogenetic fungus volatile oil in the claim 3: fresh mycelia is put into water-distillation unit, carry out the water distillation, collect volatile oil.In profit blended effluent liquid, add a certain amount of sodium-chlor, extract,, obtain volatile oil then with concentrated extract behind the anhydrous sodium sulfate drying with ether.
5, the volatile oil in the claim 3 is used to prepare antibacterial medicine, and affiliated bacterium comprises: subtilis, staphylococcus haemolyticus, angular leaf spot of cucumber bacterium, Agrobacterium tumefaciens, intestinal bacteria and tomato bacterial spot germ.
6, the volatile oil in the claim 3 is used to prepare antifungal medicament, and affiliated fungi comprises: Pyricularia oryzae.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2008101120683A CN101270340A (en) | 2008-05-21 | 2008-05-21 | Paris polyphylla var.yunnanensis endogenetic pichia for preparing volatile oil and antibiotic activity |
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| CNA2008101120683A CN101270340A (en) | 2008-05-21 | 2008-05-21 | Paris polyphylla var.yunnanensis endogenetic pichia for preparing volatile oil and antibiotic activity |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242071A (en) * | 2011-03-28 | 2011-11-16 | 国家海洋局第三海洋研究所 | Application of Pichia guilliermondii 510-6jm in treatment of petroleum hydrocarbon contaminations |
| CN110663709A (en) * | 2019-11-15 | 2020-01-10 | 中南民族大学 | Preparation method and application of paris polyphylla extractive solution |
-
2008
- 2008-05-21 CN CNA2008101120683A patent/CN101270340A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242071A (en) * | 2011-03-28 | 2011-11-16 | 国家海洋局第三海洋研究所 | Application of Pichia guilliermondii 510-6jm in treatment of petroleum hydrocarbon contaminations |
| CN102242071B (en) * | 2011-03-28 | 2013-08-14 | 国家海洋局第三海洋研究所 | Application of Pichia guilliermondii 510-6jm in treatment of petroleum hydrocarbon contaminations |
| CN110663709A (en) * | 2019-11-15 | 2020-01-10 | 中南民族大学 | Preparation method and application of paris polyphylla extractive solution |
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