CN102839148A - Fluid medium made from celery and suitable for phytophthotacactorum to generate sporangium - Google Patents
Fluid medium made from celery and suitable for phytophthotacactorum to generate sporangium Download PDFInfo
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- CN102839148A CN102839148A CN2012103231988A CN201210323198A CN102839148A CN 102839148 A CN102839148 A CN 102839148A CN 2012103231988 A CN2012103231988 A CN 2012103231988A CN 201210323198 A CN201210323198 A CN 201210323198A CN 102839148 A CN102839148 A CN 102839148A
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Abstract
The invention discloses a fluid medium made from celery and suitable for phytophthotacactorum to generate sporangium. A preparation method of the fluid medium particularly comprises the following steps: removing leaves, washing and cutting up celery, adding deionized water to celery in a w/v ratio being 1:1, and juicing by using a juicer; filtering the obtained celery juice by using four layers of gauze, diluting with deionized water containing 0.02% (w/v) CaCO3 till the celery juice is 10%-50%(v/v); and subpackaging and autoclaving for 20min at 121 DEG C under 0.103MPa; pouring autoclaved fluid medium into culture dishes with diameter being 9cm, wherein 15ml of autoclaved fluid medium into each culture dish. The raw material of the fluid medium are low in cost and can be acquired easily, the steps for making the fluid medium and generating sporangium are simple and easy to learn, and the sporangium generation efficiency of phytophthotacactorum is improved greatly, so great convenience is provided for preparation of pathogenic bacteria inoculants.
Description
Technical field
The present invention relates to that a kind of phytopathogen Phytophthora cactorum bacterium (Phytophthora cactorum) produces sporangial liquid nutrient medium 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 the host when next year, condition was suitable, 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 all need prepare sporangia suspension when learning characteristic research 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 used test materials in implementation process is various, and testing sequence is more, complicated operation, and the sporocyst that in some test liquid body substratum, produces is not easy to discharge zoospore, influences carrying out of next step test.Therefore, press for a kind of a kind of raw material substratum simple, easy and simple to handle that filters out, make Phytophthora cactorum in this liquid nutrient medium, produce a large amount of sporocysts and to be prone to discharge zoospore 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 liquid nutrient medium.The present invention has overcome and has utilized at present Phytophthora cactorum bacterium mycelia water culture to induce to produce that the sporocyst raw material is various, the shortcoming of complicated operation, provides and has cultivated the sporangial method that produces in a kind of simple and easy to do liquid medium within.
Technical scheme of the present invention is to utilize celery (Apium graveolens) to make liquid nutrient medium for raw material to be used for Phytophthora cactorum generation sporocyst.
With the celery is that the suitable Phytophthora cactorum bacterium of raw material making produces sporangial liquid nutrient medium; Concrete making method does; It is that 1: 1 ratio adds deionized water and in juicer, squeezes the juice in w/v that the celery defoliation is cleaned the chopping back, with the gained Sucus Oenanthes Javanicae with four layers of filtered through gauze after with containing 0.02% (w/v) CaCO
3Deionized water is diluted to and contains Sucus Oenanthes Javanicae 10%～50% (v/v), after the packing with 0.103MPa, 121 ℃, autoclaving 20min; The sterilising liq substratum is poured in the 9cm diameter petridish of sterilization, every ware falls 15ml.Nutrient solution 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 in the liquid nutrient medium, and every ware is put into 5 mycelia pieces.After postvaccinal petridish placed 25 ℃ of following dark culturing 5d, liquid nutrient medium is outwelled, after continuing to cultivate 2d with sterilized water, changed one time sterilized water, just produce a large amount of sporocysts after cultivating 3d again.Place 4 ℃ of refrigerators to handle 30-60min in petridish subsequently, place 25 ℃ to cultivate 1-2h down again, sporocyst begins to discharge in a large number zoospore.
The invention has the beneficial effects as follows: the liquid nutrient medium that utilizes a kind of raw material of celery to make just can produce a large amount of sporocysts, has overcome to adopt 2 kinds or the mixing of various vegetables different ratios just can obtain sporangial numerous and diverse operations in a large number in the past.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 evaluation of sporocyst form, the relevant researchs such as preparation that Phytophthora cactorum bacterium virulence is measured zoospore suspension-s, has higher actual application value.
Embodiment
Below in conjunction with specific embodiment, the present invention is elaborated.
Embodiment: celery liquid nutrient medium and other liquid nutrient medium product sporocyst ability and zoospore releasability diversity ratio are
1, liquid culture matrix manufacturing:
(1) cucumber substratum (10%): cucumber is cleaned the chopping back and in juicer, squeezes the juice, gained juice with four layers of filtered through gauze after and contain 0.02%CaCO
31: 9 by volume ratio mixing of deionized water.
(2) cucumber substratum (50%): cucumber is cleaned the chopping back and in juicer, squeezes the juice, gained juice with four layers of filtered through gauze after and contain 0.02%CaCO
31: 1 by volume ratio mixing of deionized water.
(3) tomato substratum (10%): tomato is cleaned the chopping back and in juicer, squeezes the juice, gained juice with four layers of filtered through gauze after and contain 0.02%CaCO
31: 9 by volume ratio mixing of deionized water.
(4) tomato substratum (50%): tomato is cleaned the chopping back and in juicer, squeezes the juice, gained juice with four layers of filtered through gauze after and contain 0.02%CaCO
31: 1 by volume ratio mixing of deionized water.
(5) celery substratum (10%): celery is cleaned the chopping back and in juicer, is squeezed the juice, gained juice with four layers of filtered through gauze after and contain 0.02%CaCO
31: 9 by volume ratio mixing of deionized water.
(6) celery substratum (25%): celery is cleaned the chopping back and in juicer, is squeezed the juice, gained juice with four layers of filtered through gauze after and contain 0.02%CaCO
31: 3 by volume ratio mixing of deionized water.
(7) celery substratum (50%): celery is cleaned the chopping back and in juicer, is squeezed the juice, gained juice with four layers of filtered through gauze after and contain 0.02%CaCO
31: 1 by volume ratio mixing of deionized water.
(8) tomato: cucumber substratum (10%): tomato and cucumber are cleaned the chopping back in juicer, squeeze the juice, gained juice is pressed tomato and 1: 2 mixed of cucumber volume ratio with four layers of filtered through gauze, again with mixed solution with containing 0.02%CaCO
3Deionized water is diluted to 10%.
(9) tomato: cucumber substratum (50%): tomato and cucumber are cleaned the chopping back in juicer, squeeze the juice, gained juice is pressed tomato and 1: 2 mixed of cucumber volume ratio with four layers of filtered through gauze, again with mixed solution with containing 0.02%CaCO
3Deionized water is diluted to 50%.
(10) tomato: celery substratum (10%): tomato and celery are cleaned the chopping back in juicer, squeeze the juice, gained juice is pressed tomato and 1: 2 mixed of celery volume ratio with four layers of filtered through gauze, again with mixed solution with containing 0.02%CaCO
3Deionized water is diluted to 10%.
(11) tomato: celery substratum (50%): tomato and celery are cleaned the chopping back in juicer, squeeze the juice, gained juice is pressed tomato and 1: 2 mixed of celery volume ratio with four layers of filtered through gauze, again with mixed solution with containing 0.02%CaCO
3Deionized water is diluted to 50%.
With above-mentioned substratum respectively after the packing in 0.103MPa, 121 ℃, autoclaving 20min.Be cooled to about 40 ℃ and make liquid nutrient medium.
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 above-mentioned 11 kinds of different liqs substratum, every ware is put into 5 mycelia pieces.Postvaccinal petridish is placed 25 ℃ of following dark culturing 5d, liquid nutrient medium is outwelled, after continuing to cultivate 2d with sterilized water, change one time sterilized water, cultivate 3d " Invest, Then Investigate " liquid nutrient medium miospore capsule generation again.Each substratum repeats 3 times.Investigate from petridish at every turn and get five 1cm with five-spot
2The mycelia piece of size, 10 times of eyepieces of microscope are counted the sporocyst quantity that produces on each visual field mycelia piece down, and statistical study different liqs substratum produces the difference of sporocyst ability.
After treating that the Phytophthora cactorum bacterium produces sporocyst in a large number in the different liqs substratum; Petridish is placed 4 ℃ of refrigerator 30-60min; Take out again and place 25 ℃ to cultivate down; Observe the ratio that zoospore discharges in the counting different liqs substratum, the difference of zoospore releasability in the statistical study different liqs substratum.
The Phytophthora cactorum bacterium is cultivated the ability that produces sporocyst and zoospore release and supplies examination substratum (table 1) apparently higher than other in the celery liquid nutrient medium.In addition, the concentration difference of Sucus Oenanthes Javanicae is also influential to sporulation quantity and zoospore burst size in the celery liquid nutrient medium, celery substratum (10%)＞celery substratum (25%)＞celery substratum (50%).
Table 1 celery substratum and other substratum produce the sporocyst capacity variance relatively
| Supply the examination substratum | Unit surface produces sporangial amount (individual/visual field) | Zoospore discharges ratio (%) |
| Cucumber substratum (10%) | 17±7F | 52.6 |
| Cucumber substratum (50%) | 83±2D | 12.8 |
| Tomato substratum (10%) | 88±4D | 57.5 |
| Tomato substratum (50%) | 113±19C | 8.3 |
| Celery substratum (10%) | 214±10A | 92.3 |
| Celery substratum (25%) | 196±7AB | 87.9 |
| Celery substratum (50%) | 174±5B | 85.4 |
| Tomato: cucumber substratum (10%) | 111±7C | 61.8 |
| Tomato: cucumber substratum (50%) | 11±3F | 26.7 |
| Tomato: celery substratum (10%) | 17±2F | 71.6 |
| Tomato: celery substratum (50%) | 44±4E | 13.5 |
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. one kind is that the suitable Phytophthora cactorum bacterium of raw material making produces sporangial liquid nutrient medium with the celery; It is characterized in that; Concrete making method does; It is that 1: 1 ratio adds deionized water and in juicer, squeezes the juice in w/v that the celery defoliation is cleaned the chopping back, with gained juice with four layers of filtered through gauze after with containing 0.2% (w/v) CaCO
3Deionized water be diluted to and contain Sucus Oenanthes Javanicae 10%～50% (v/v), after the packing with 0.103MPa, 121 ℃, autoclaving 20min; Sterilising medium is poured in the petridish of sterilization and processed liquid nutrient medium.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210323198.8A CN102839148B (en) | 2012-08-28 | 2012-08-28 | Fluid medium made from celery and suitable for phytophthotacactorum to generate sporangium |
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| CN201210323198.8A CN102839148B (en) | 2012-08-28 | 2012-08-28 | Fluid medium made from celery and suitable for phytophthotacactorum to generate sporangium |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104726349A (en) * | 2015-04-15 | 2015-06-24 | 云南省烟草农业科学研究院 | Tobacco phytophthora culture medium and preparation method thereof |
| CN108410793A (en) * | 2018-04-27 | 2018-08-17 | 江西省农业科学院植物保护研究所 | A kind of induction phytophthora blight of pepper production culture medium of spore, preparation method and application |
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| CN102154193A (en) * | 2011-01-20 | 2011-08-17 | 天津市植物保护研究所 | Method for inducing Phytophthora capsici to produce large amount of sporangia |
| CN102643776A (en) * | 2012-03-31 | 2012-08-22 | 云南农业大学 | Method for preparing culture medium appropriate for phytophthotacactorum to produce sporangiums by taking radish as raw material |
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Patent Citations (3)
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| CN102154193A (en) * | 2011-01-20 | 2011-08-17 | 天津市植物保护研究所 | Method for inducing Phytophthora capsici to produce large amount of sporangia |
| CN102643776A (en) * | 2012-03-31 | 2012-08-22 | 云南农业大学 | Method for preparing culture medium appropriate for phytophthotacactorum to produce sporangiums by taking radish as raw material |
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| 《农药学学报》 20120430 邓维萍等 胶孢炭疽菌及两种疫霉菌对苯酰菌胺的敏感性与beta-微管蛋白氨基酸突变的相关性分析 全文 1 第14卷, 第2期 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104726349A (en) * | 2015-04-15 | 2015-06-24 | 云南省烟草农业科学研究院 | Tobacco phytophthora culture medium and preparation method thereof |
| CN104726349B (en) * | 2015-04-15 | 2018-05-25 | 云南省烟草农业科学研究院 | A kind of Phytophthora nicotianae culture medium and preparation method thereof |
| CN108410793A (en) * | 2018-04-27 | 2018-08-17 | 江西省农业科学院植物保护研究所 | A kind of induction phytophthora blight of pepper production culture medium of spore, preparation method and application |
| CN108410793B (en) * | 2018-04-27 | 2020-11-06 | 江西省农业科学院植物保护研究所 | Culture medium for inducing phytophthora capsici to produce spores, preparation method and application |
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