CN102643776A - Method for preparing culture medium appropriate for phytophthotacactorum to produce sporangiums by taking radish as raw material - Google Patents
Method for preparing culture medium appropriate for phytophthotacactorum to produce sporangiums by taking radish as raw material Download PDFInfo
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- CN102643776A CN102643776A CN2012100912535A CN201210091253A CN102643776A CN 102643776 A CN102643776 A CN 102643776A CN 2012100912535 A CN2012100912535 A CN 2012100912535A CN 201210091253 A CN201210091253 A CN 201210091253A CN 102643776 A CN102643776 A CN 102643776A
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Abstract
The invention discloses a method for preparing a culture medium appropriate for phytophthotacactorum to produce sporangiums by taking radish as a raw material. The concrete preparation method comprises the following steps of: taking 100-200g of radish; chopping and boiling in 0.5 L of water for 30 minutes; filtering; adding 15g of agar in filtrate, heating to melt the agar with the constant volume to be 1 L; split charging; carrying out steam sterilization at high pressure of 0.103MPa and 121 DEG C for 20 minutes; putting the sterilized culture medium into a sterilized culture dish with the diameter of 9cm so as to form a surface plate; after cooling, punching the edge of a bacterial colony cultured for 3 days through a puncher with the diameter being 5mm to obtain bacterial blocks; inoculating the bacterial blocks on the culture medium; and culturing the inoculated culture dish in darkness for 10 days at 25 DEG C so as to produce a large number of sporangiums. The probability of pollution in an operation process that a water culture method is utilized for inducing to produce the sporangiums is reduced. The raw material of the culture medium is low in cost and is easy to obtain, and the processes of preparing the culture medium and producing the sporangiums are simple and easy to learn.
Description
Technical field
The present invention relates to that a kind of phytopathogen Phytophthora cactorum bacterium (Phytophthora cactorum) produces sporangial substratum in the plant protection field.
Background technology
Phytophthora cactorum (Phytophthora cactorum Schroet) belongs to oomycetes door phytophthora pathogenic bacteria, is the important phytopathogen that extensively distributes in the worldwide, can infect about 200 kind of plant in about 60 sections, 50 genus.The Phytophthora cactorum bacterium survives the winter in invalid body and soil with mycelium and oospore usually.Mycelia was directly infected and posts when next year, condition was fit to, or formed a large amount of zoosporangiums and propagate into overground part and infect cauline leaf.Sporocyst plays important effect in the propagation of this disease with in spreading.Usually, carry out that virulence is measured or other associated biomolecule is learned characteristic research and all need be prepared spore suspension indoor.At present; Induce Phytophthora cactorum to produce sporangial method and mainly adopt the mycelia water culture; Main mode has (1) that cultured mycelium is placed the Pi Shi nutrient solution that disinfection soil leach liquor or sterile purified water are housed, and petridish is placed luminescent lamp under shone 3 days again, and ware liquid 3 times vibrates every day; Liquid make-up for the third time, and cultivate down at 8 ℃ zoospore is discharged.(2) cut the petridish that the mycelia piece is put into 15ml 10%V6 nutrient solution from colony edge, place 25 ℃, dark to cultivate 3d, then mycelia is chosen in the petridish of 15ml sterile purified water, place 25 ℃ of following illumination 3d, change water every day 1 time.After will filling a large amount of sporangial petridish after sporocyst produces and putting into 4 ℃ of refrigerator 15min, move under 25 ℃ and leave standstill 20min, lure that a large amount of zoospores produce into.Various water cultures testing sequence in implementation process is more, and misoperation will be influenced the test process by living contaminants a little.Therefore, press for a kind of a kind of substratum that filters out, make Phytophthora cactorum on this substratum, produce a large amount of sporocysts and be used for correlative study.
Summary of the invention
The purpose of this invention is to provide and a kind ofly make the Phytophthora cactorum bacterium produce sporangial substratum.The present invention has overcome and utilizes Phytophthora cactorum bacterium mycelia water culture to induce at present to produce sporocyst easily by the shortcoming of living contaminants, a kind of sporangial method that produces of directly on substratum, cultivating is provided.
Technical scheme of the present invention is to utilize radish (Raphanus sativus L.) to make substratum for raw material to be used for Phytophthora cactorum generation sporocyst.
With the radish is that the suitable Phytophthora cactorum bacterium of raw material making produces sporangial substratum; Concrete making method does; Get 100-200 gram radish, filtration in 30 minutes is boiled in the chopping back in 0.5L water, adds 15g agar in the filtrating; Heating is settled to 1 liter after agar is melted, after the packing with 0.103MPa, 121C, autoclaving 20min; Sterilising medium is processed flat board in the 9cm diameter petridish of going into sterilization, the cooling back is beaten at the colony edge of cultivating 3d with the punch tool of diameter 5mm and is got the bacterium piece and be inoculated on the substratum.Place 25 ℃ of following dark culturing just to produce a large amount of sporocysts after 10 days in postvaccinal petridish.
The invention has the beneficial effects as follows: directly on the radish substratum, just cultivate and to produce a large amount of sporocysts, reduced and utilized water culture to induce the probability that pollutes in the sporangial operating process of generation.In addition; This substratum material cost is cheap and obtain easily; The program that the making of substratum and sporocyst produce is easy to learn; And this method can be used for the relevant researchs such as preparation of evaluation, Phytophthora cactorum bacterium virulence mensuration or the resistance screening mutant miospore suspension-s of sporocyst form, has higher actual application value.
Embodiment
Below in conjunction with specific embodiment, the present invention is elaborated.
Embodiment one: radish nutrient agar and other substratum produce the sporocyst capacity variance relatively
1, substratum is made:
(1) radish substratum (100g/L): get the 100g radish, filtration in 30 minutes is boiled in the chopping back in 0.5L water, adds the 15g agar powder in the filtrating, and heating is settled to 1 liter after agar is melted.
(2) radish substratum (150g/L): get the 150g radish, filtration in 30 minutes is boiled in the chopping back in 0.5L water, adds the 15g agar powder in the filtrating, and heating makes fixed the dissolving to 1 liter in agar thawing back.
(3) radish substratum (200g/L): get the 200g radish, filtration in 30 minutes is boiled in the chopping back in 0.5L water, adds the 15g agar powder in the filtrating, and heating makes fixed the dissolving to 1 liter in agar thawing back.
(4) Semen Phaseoli Vulgaris substratum: white clouds bean powder 60g, in 0.5L water, boil filtration in 30 minutes, add the 15g agar powder in the filtrating, heating makes agar melt back zero(ppm) water and is settled to 1L.
(5) Radix Dauci Sativae substratum (CA) substratum: get Radix Dauci Sativae 200g, in 0.5L water, boil filtration in 30 minutes, add the 15g agar powder in the filtrating, heating makes agar melt back zero(ppm) water and is settled to 1L.
(6) potato dextrose agar (PDA): get yam 200g, in 0.5L water, boil filtration in 30 minutes, add glucose 6g, agar powder 15g in the filtrating, zero(ppm) water is settled to 1L.
With above-mentioned substratum respectively after the packing in 0.103MPa, 121 ℃, autoclaving 20min.Be cooled to about 50 ℃ and make culture medium flat plate.
2, different substratum produce spore capacity variance mensuration
Beat at the colony edge of cultivating 3d with the punch tool of diameter 5mm and to get the bacterium piece, place on above-mentioned 6 kinds of different culture medium flat plates 25 ℃ to cultivate dark culturing down down, cultivate sporocyst generation on back 5d, 10d, 13d, 15d and the 18d investigation substratum.Each substratum triplicate.Investigate from petridish at every turn and get five 1cm with five-spot
2The substratum of size, the sporocyst quantity that produces on every square centimeter of substratum of microscopically counting, the different substratum of statistical study produce the difference of sporocyst ability.
The Phytophthora cactorum bacterium is directly cultivated the sporangial ability of product and is significantly higher than Radix Dauci Sativae, yam and Semen Phaseoli Vulgaris substratum (table 1) on the radish substratum.In addition, the radish of different amounts is also influential to sporulation quantity in the radish substratum, its sporulation quantity radish substratum (150g/L)＞radish substratum (200g/L)＞radish substratum (100g/L).
Table 1 radish substratum and other substratum commonly used produce the sporocyst capacity variance relatively
* represent The results of analysis of variance (p＞0.05), same letter shows no significant difference.
Embodiment two: measure the Phytophthora cactorum bacterium to the zoospore suspension preparation in the pseudo-ginseng blade virulence
1, substratum is made: get 200 gram radish, filtrations in 30 minutes boiled in the chopping back in 0.5L water, adds 15g agar in the filtrating, and it is fixed molten to 1 liter that heating makes agar melt the back, after the packing with 0.103MPa, 121 ℃, autoclaving 20min.Be cooled to about 50 ℃ and make culture medium flat plate.
2, the preparation of spore suspension: beat at the colony edge of cultivating 3d with the punch tool of diameter 5mm and to get the bacterium piece; Place 25 ℃ of cultivations down on the culture medium flat plate; In each petridish, add the release of 4 ℃ of sterilized waters stimulations of 10mL sporocyst after waiting to grow a large amount of sporocysts, zoospore discharges after about 30-60 minute.With pipettor spore suspension is transferred in the beaker, blood counting chamber is measured its concentration, and ultimate density is adjusted into every milliliter contains and have an appointment 10
5The spore suspension of individual zoospore.
3, virulence is measured: utilize sessile drop method to be inoculated in the pseudo-ginseng face of blade of exsomatizing spore suspension; Each blade inoculation 20 μ l; Place the diameter 15cm petridish of preserving moisture to cultivate 12h down in blade after the inoculation,, cultivate greater than 80%, under the condition of 12h alternation of light and darkness in 23 ℃, humidity then to guarantee the intrusion of pathogenic bacteria in 20 ℃ of dark conditions with thieving paper; Can observe the inoculation point behind the 3d and begin to occur the water soaking mode scab, on scab, occur the mould layer of white behind the 5d.
Embodiment three: the evaluation of Phytophthora cactorum bacterium sporocyst form
1, substratum is made: get 100 gram radish, be cut into small pieces and in 0.5L water, boil 30 minutes after-filtration, add 15g agar in the filtrating, heating is settled to 1 liter after agar is melted, after the packing with 0.103MPa, 121 ℃, autoclaving 20min.Be cooled to about 50 ℃ to culture medium flat plate.
2, bacterium colony and sporocyst form are identified: beat at the colony edge of cultivating 3d with the punch tool of diameter 5mm and get the bacterium piece; Place 25 ℃ of cultivations down on the different culture medium flat plates; 7d observes the form and the color of bacterium colony; Sporangial shape, size, long-width ratio, top structure (having or not and thickness of mastoid process), sporocyst come off proterties and come off the back sporangiophore length.The result shows that on the radish substratum, the Phytophthora cactorum primary hyphae is colourless, and mycelia almost can't see aerial hyphae along growing on the substratum.It is radial that bacterium colony is polygon, and will approach on the growth phase contrast PDA substratum is a lot, but sporocyst can produce in a large number, and this is very beneficial for the observation of sporocyst form.Microscopically is observed and is shown, give birth on the sporocyst top, spherical or avette, and base portion is circular, has a tangible mastoid process; Sporangiophore is short, is prone to after the maturation come off.Phytophthora cactorum (Phytophthora cactorum) basically identical that these characteristics and Newhook (1978) and surplus Yongnian (1986) describe.
Embodiment four: carry out the resistance mutant choice with Phytophthora cactorum bacterium spore suspension
1, substratum is made: get 150 gram radish, be cut into small pieces and in 0.5L water, boil 30 minutes after-filtration, add 15g agar in the filtrating, heating makes agar melt that the back is fixed dissolves to 1 liter, after the packing with 0.103MPa, 121 ℃, autoclaving 20min.Be cooled to about 50 ℃ and make culture medium flat plate.
2, the preparation of spore suspension: beat at the colony edge of cultivating 3d with the punch tool of diameter 5mm and to get the bacterium piece; Place 25 ℃ of cultivations down on the different culture medium flat plates; In each petridish, add the release of 4 ℃ of sterilized waters stimulations of 10mL sporocyst after waiting to grow a large amount of sporocysts, zoospore discharges after about 30-60 minute.With pipettor spore suspension is transferred in the beaker, blood counting chamber is measured its concentration, and ultimate density is adjusted into every milliliter contains and have an appointment 10
5The spore suspension of individual zoospore.
3, the direct screening of resistance two mutants: zoospore suspension-s is coated on the radish that contains SYP-L190 (10 μ g/mL) cultivates on the base band poison flat board, it is 10 that every ware is coated with 100 μ L concentration
6The spore suspension of individual zoospore/mL is coated with 50 wares altogether.Phytophthora cactorum bacteria growing situation on the malicious substratum is with in inspection after cultivating 5d under 25 ℃ of dark conditions.The result shows, grows 3 bacterium colonies on 50 wares altogether, and its resistance frequency is 6 * 10
-8
4, the ultraviolet induction of drug-fast strain: (every milliliter contains 10 with 5mL zoospore suspension-s
6Individual zoospore) adds in the petridish; Behind 20cm distance irradiation 1min (it is 10-30% that this dosage is handled back zoospore survival rate) under the UV-lamp of 20W, 254nm, will place through the suspension-s that uviolizing is handled immediately and cultivate 30min under the dark condition, to reduce the light reparation.Subsequently spore suspension is coated the white turnip that contains SYP-L190 (10 μ g/mL) and cultivate on the base band poison flat board every ware 100 μ l, totally 50 wares.Test is a blank with the zoospore suspension-s without UV treatment.After cultivating 5d under 25 ℃ of dark conditions, from 50 wares, grow 6 bacterium colonies, its resistance frequency is 1.2 * 10
-7
Should be understood that, concerning those of ordinary skills, can improve or conversion, and all these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention according to above-mentioned explanation.
Claims (1)
1. a radish is that the suitable Phytophthora cactorum bacterium of raw material making produces sporangial substratum, it is characterized in that concrete making method does; Get 100-200 gram radish; Boiled in 0.5L water 30 minutes the chopping back, filters, and adds 15g agar in the filtrating; Heating is settled to 1 liter after agar is melted, after the packing with 0.103MPa, 121C, autoclaving 20min; Sterilising medium is poured in the petridish of sterilization and processed flat board.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839148A (en) * | 2012-08-28 | 2012-12-26 | 云南农业大学 | Fluid medium made from celery and suitable for phytophthotacactorum to generate sporangium |
| CN102839147A (en) * | 2012-08-28 | 2012-12-26 | 云南农业大学 | Fluid medium made from pseudo-ginseng and suitable for phytophthotacactorum to generate zoospore |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810952A (en) * | 2006-02-16 | 2006-08-02 | 苏州农业职业技术学院 | Microbe fermenting culture liquid and its prepn process |
| CN102226167A (en) * | 2011-05-31 | 2011-10-26 | 云南省烟草农业科学研究院 | Method for inducing phytophthora parasitica var. nicotianae to generate zoosporangium and release spore |
| CN102321711A (en) * | 2011-08-31 | 2012-01-18 | 红云红河烟草(集团)有限责任公司 | Method for inducing phytophthora nicotianae to generate pathogenic secretory protein |
| CN102391980A (en) * | 2011-11-25 | 2012-03-28 | 福建省农业科学院植物保护研究所 | Simple and effective method for producing zoospores through inducing Phytophthora capsici |
-
2012
- 2012-03-31 CN CN 201210091253 patent/CN102643776B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810952A (en) * | 2006-02-16 | 2006-08-02 | 苏州农业职业技术学院 | Microbe fermenting culture liquid and its prepn process |
| CN102226167A (en) * | 2011-05-31 | 2011-10-26 | 云南省烟草农业科学研究院 | Method for inducing phytophthora parasitica var. nicotianae to generate zoosporangium and release spore |
| CN102321711A (en) * | 2011-08-31 | 2012-01-18 | 红云红河烟草(集团)有限责任公司 | Method for inducing phytophthora nicotianae to generate pathogenic secretory protein |
| CN102391980A (en) * | 2011-11-25 | 2012-03-28 | 福建省农业科学院植物保护研究所 | Simple and effective method for producing zoospores through inducing Phytophthora capsici |
Non-Patent Citations (4)
| Title |
|---|
| C. L. SCHEIDFR AND D. L. YODER: "Development of a Methodology for the Production of Aphanomyces cochlioides Oospores in Vitro", 《JOURNAL OF THE A.S.S.B.T》 * |
| 张俊华等: "南瓜疫病菌(Phytophthora capsici)的生物学特性初探", 《东北农业大学学报》 * |
| 徐莹等: "牡蛎萝卜汁的乳酸菌发酵研究", 《中国酿造》 * |
| 赵雁等: "梨树恶疫霉的生物学特性研究", 《中国果树》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839148A (en) * | 2012-08-28 | 2012-12-26 | 云南农业大学 | Fluid medium made from celery and suitable for phytophthotacactorum to generate sporangium |
| CN102839147A (en) * | 2012-08-28 | 2012-12-26 | 云南农业大学 | Fluid medium made from pseudo-ginseng and suitable for phytophthotacactorum to generate zoospore |
| CN102839147B (en) * | 2012-08-28 | 2014-12-17 | 云南农业大学 | Fluid medium made from pseudo-ginseng and suitable for phytophthotacactorum to generate zoospore |
| CN102839148B (en) * | 2012-08-28 | 2014-12-17 | 云南农业大学 | Fluid medium made from celery and suitable for phytophthotacactorum to generate sporangium |
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