CN102643776B - Culture medium appropriate for phytophthotacactorum to produce sporangiums by taking radish as raw material - Google Patents
Culture medium appropriate for phytophthotacactorum to produce sporangiums by taking radish as raw material Download PDFInfo
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- CN102643776B CN102643776B CN 201210091253 CN201210091253A CN102643776B CN 102643776 B CN102643776 B CN 102643776B CN 201210091253 CN201210091253 CN 201210091253 CN 201210091253 A CN201210091253 A CN 201210091253A CN 102643776 B CN102643776 B CN 102643776B
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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 needed to prepare spore suspension indoor.At present, induce Phytophthora cactorum to produce sporangial method and mainly adopt the mycelia water culture, main mode has (1) cultured mycelium to be placed the Pi Shi nutrient solution that disinfection soil leach liquor or sterile purified water are housed, again culture dish is placed under the luminescent lamp and shone 3 days, 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 culture dish 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 culture dish 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 culture dish 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 produce a large amount of sporocysts at this substratum 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, provide a kind of and directly cultivated the sporangial method that produces at substratum.
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.
Be that raw material is made suitable Phytophthora cactorum bacterium and produced sporangial substratum with the radish, concrete making method is, get 100-200 gram radish, filtration in 30 minutes is boiled in the chopping back in 0.5L water, add 15g agar in the filtrate, heating is settled to 1 liter after agar is melted, after the packing with 0.103MPa, 121C, autoclaving 20min; Sterilising medium is made flat board in the 9cm diameter culture dish 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 culture dish.
The invention has the beneficial effects as follows: directly just cultivate at the radish substratum and can 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 of sporocyst form, has higher actual application value.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
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 filtrate, 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 filtrate, and heating makes agar thawing back fixed molten to 1 liter.
(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 filtrate, and heating makes agar thawing back fixed molten to 1 liter.
(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 filtrate, heating makes agar melt back distilled water and is settled to 1L.
(5) Radix Dauci Sativae substratum (CA) substratum: get Radix Dauci Sativae 200g, boil filtration in 30 minutes in 0.5L water, add the 15g agar powder in the filtrate, heating makes agar melt back distilled water and is settled to 1L.
(6) potato dextrose agar (PDA): get potato 200g, boil filtration in 30 minutes in 0.5L water, add glucose 6g, agar powder 15g in the filtrate, distilled 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 culture dish 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, potato 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 filtrate, heating make agar melt the back decide molten 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 culture medium flat plate, add the release of 4 ℃ of sterilized waters stimulations of 10mL sporocyst after waiting to grow a large amount of sporocysts in each culture dish, 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 culture dish of preserving moisture with thieving paper under 20 ℃ of dark conditions, to cultivate 12h in blade after the inoculation, to guarantee the intrusion of pathogenic bacteria, cultivate greater than 80%, under the condition of 12h alternation of light and darkness in 23 ℃, humidity then, can observe the inoculation point behind the 3d and begin to occur the water soaking mode scab, the mould layer of white occur at scab 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 boil 30 minutes after-filtration in 0.5L water, add 15g agar in the filtrate, 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 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 be can't see aerial hyphae along growing on the substratum.It is radial that bacterium colony is polygon, and wanting on the growth phase contrast PDA substratum is thin 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, and spherical or avette, the base portion circle has a tangible mastoid process; Sporangiophore is short, easily comes off after the maturation.Phytophthora cactorum (Phytophthora cactorum) basically identical that these features 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 boil 30 minutes after-filtration in 0.5L water, add 15g agar in the filtrate, 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 different culture medium flat plates, add the release of 4 ℃ of sterilized waters stimulations of 10mL sporocyst after waiting to grow a large amount of sporocysts in each culture dish, 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 mutant: zoospore suspension is coated on the radish that contains flumorph (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.After cultivating 5d under 25 ℃ of dark conditions, check the Phytophthora cactorum bacteria growing situation on the malicious substratum of being with.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
6Individual zoospore) adds in the culture dish, 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 under the dark condition through the suspension that uviolizing is handled immediately and cultivate 30min, to reduce the light reparation.Subsequently spore suspension is coated the white turnip that contains flumorph (10 μ g/mL) and cultivated on the base band poison flat board every ware 100 μ l, totally 50 wares.Test is blank with the zoospore suspension 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, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (1)
1. radish produces application in the sporangial substratum making suitable Phytophthora cactorum bacterium, it is characterized in that, concrete grammar is, get 100-200 gram radish, boiled in 0.5L water 30 minutes the chopping back, filters, and adds 15g agar in the filtrate, heating is settled to 1 liter after agar is melted, after the packing with 0.103MPa, 121 ℃, autoclaving 20min; Sterilising medium is poured in the culture dish of sterilization and made flat board.
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| 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 |
| 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 |
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