CN102839148B - 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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- CN102839148B CN102839148B CN201210323198.8A CN201210323198A CN102839148B CN 102839148 B CN102839148 B CN 102839148B CN 201210323198 A CN201210323198 A CN 201210323198A CN 102839148 B CN102839148 B CN 102839148B
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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 a kind of phytopathogen Phytophthora cactorum bacterium (Phytophthora cactorum) in plant protection art and produce sporangial liquid nutrient medium.
Background technology
Phytophthora cactorum (Phytophthora cactorum Schroet), belonging to oomycetes door phytophthora pathogenic bacteria, is the important phytopathogen of extensively distribution in worldwide, can infect about 200 kind of plant in about 60 sections, 50 genus.Phytophthora cactorum bacterium survives the winter in invalid body and soil with mycelium and oospore usually.When next year, condition was suitable for, mycelia directly infects host, or forms a large amount of zoosporangium and propagate into overground part and infect cauline leaf.Sporocyst is in the propagation of this disease and play very important effect in spreading.Usually, all need to prepare sporangia suspension when carrying out Pathogenic Tests or other related biological characteristic research in indoor.At present, induction Phytophthora cactorum produces sporangial method and mainly adopts mycelia water culture, major way has (1) is placed in the Pi Shi nutrient solution that disinfection soil leach liquor or sterile purified water are housed by cultured mycelium, irradiate 3 days under again culture dish being placed in luminescent lamp, vibrate every day ware liquid 3 times, liquid make-up for the third time, and cultivation makes Zoospore liberation in 2 hours at 8 DEG C.(2) cut from colony edge the culture dish that mycelia block puts into 15ml 10%V6 nutrient solution, be placed in 25 DEG C, dark cultivates 3d, then mycelia is chosen in the culture dish of 15ml sterile purified water, illumination 3d at being placed in 25 DEG C, change water every day 1 time.Sporocyst will fill after a large amount of sporangial culture dish puts into 4 DEG C of refrigerator 15min after producing, leave standstill 20min, lure that a large amount of zoospore produces at moving to 25 DEG C.Various water culture test materials used in implementation process is various, and testing sequence is more, complicated operation, and the sporocyst produced in some test liquid body substratum is not easy to discharge zoospore, affects carrying out of next step test.Therefore, filter out a kind of raw material substratum simple, easy and simple to handle in the urgent need to a kind of, make Phytophthora cactorum can produce a large amount of sporocyst in this liquid nutrient medium and easily discharge zoospore for correlative study.
Summary of the invention
The object of this invention is to provide one makes Phytophthora cactorum bacterium produce sporangial liquid nutrient medium.Instant invention overcomes and utilize Phytophthora cactorum bacterium mycelia water culture to induce to produce that sporocyst raw material is various, the shortcoming of complicated operation at present, provide in a kind of simple and easy to do liquid medium within to cultivate to produce sporangial method.
Technical scheme of the present invention utilizes celery (Apium graveolens) to be used for Phytophthora cactorum generation sporocyst for raw material makes liquid nutrient medium.
Be that the suitable Phytophthora cactorum bacterium of raw material making produces sporangial liquid nutrient medium with celery, concrete making method is, the ratio being 1: 1 in w/v after celery defoliation cleans chopping adds deionized water and squeezes the juice in juice extractor, gained Sucus Oenanthes Javanicae is used containing 0.02% (w/v) CaCO with after four layers of filtered through gauze
3deionized water is diluted to containing Sucus Oenanthes Javanicae 10% ~ 50% (v/v), with 0.103MPa, 121 DEG C, autoclaving 20min after packing; Poured into by sterilising liq substratum in the 9cm diameter Petri dishes of sterilizing, every ware falls 15ml.To beat at the colony edge cultivating 3d with the punch tool of diameter 5mm after nutrient solution cooling and get bacterium block and be inoculated in liquid nutrient medium, 5 mycelia blocks put into by every ware.At postvaccinal culture dish is placed in 25 DEG C after dark culturing 5d, liquid nutrient medium is outwelled, after continuing to cultivate 2d with sterilized water, change a sterilized water, more just produce a large amount of sporocyst after cultivating 3d.Subsequently culture dish is placed in 4 DEG C of refrigerators and processes 30-60min, then cultivate 1-2h at being placed in 25 DEG C, sporocyst starts to discharge zoospore in a large number.
The invention has the beneficial effects as follows: the liquid nutrient medium utilizing a kind of raw material of celery to make just can produce a large amount of sporocyst, overcome employing 2 kinds in the past or various vegetables different ratios and mix and could obtain a large amount of sporangial numerous and diverse operation.In addition, this substratum material cost is cheap and easily obtain, the program that the making of substratum and sporocyst produce is easy to learn, and this method may be used for the relevant research such as the preparation of zoospore suspension in the qualification of sporangial morphology, Phytophthora cactorum bacterium Pathogenic Tests, has higher actual application value.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment: celery liquid nutrient medium produces sporocyst ability with other liquid nutrient medium and Zoospore liberation capacity variance compares
1, liquid culture matrix manufacturing:
(1) cucumber substratum (10%): cucumber is squeezed the juice after cleaning chopping in juice extractor, gained juice is with after four layers of filtered through gauze and containing 0.02%CaCO
3deionized water is 1: 9 ratio mixing by volume.
(2) cucumber substratum (50%): cucumber is squeezed the juice after cleaning chopping in juice extractor, gained juice is with after four layers of filtered through gauze and containing 0.02%CaCO
3deionized water is 1: 1 ratio mixing by volume.
(3) tomato substratum (10%): tomato is squeezed the juice after cleaning chopping in juice extractor, gained juice is with after four layers of filtered through gauze and containing 0.02%CaCO
3deionized water is 1: 9 ratio mixing by volume.
(4) tomato substratum (50%): tomato is squeezed the juice after cleaning chopping in juice extractor, gained juice is with after four layers of filtered through gauze and containing 0.02%CaCO
3deionized water is 1: 1 ratio mixing by volume.
(5) celery substratum (10%): celery is squeezed the juice after cleaning chopping in juice extractor, gained juice is with after four layers of filtered through gauze and containing 0.02%CaCO
3deionized water is 1: 9 ratio mixing by volume.
(6) celery substratum (25%): celery is squeezed the juice after cleaning chopping in juice extractor, gained juice is with after four layers of filtered through gauze and containing 0.02%CaCO
3deionized water is 1: 3 ratio mixing by volume.
(7) celery substratum (50%): celery is squeezed the juice after cleaning chopping in juice extractor, gained juice is with after four layers of filtered through gauze and containing 0.02%CaCO
3deionized water is 1: 1 ratio mixing by volume.
(8) tomato: cucumber substratum (10%): squeeze the juice in juice extractor after tomato and cucumber are cleaned chopping, gained juice four layers of filtered through gauze, mix in the ratio of tomato with cucumber volume ratio 1: 2, then by mixed solution with containing 0.02%CaCO
3deionized water is diluted to 10%.
(9) tomato: cucumber substratum (50%): squeeze the juice in juice extractor after tomato and cucumber are cleaned chopping, gained juice four layers of filtered through gauze, mix in the ratio of tomato with cucumber volume ratio 1: 2, then by mixed solution with containing 0.02%CaCO
3deionized water is diluted to 50%.
(10) tomato: celery substratum (10%): squeeze the juice in juice extractor after tomato and celery are cleaned chopping, gained juice four layers of filtered through gauze, mix in the ratio of tomato with celery volume ratio 1: 2, then by mixed solution with containing 0.02%CaCO
3deionized water is diluted to 10%.
(11) tomato: celery substratum (50%): squeeze the juice in juice extractor after tomato and celery are cleaned chopping, gained juice four layers of filtered through gauze, mix in the ratio of tomato with celery volume ratio 1: 2, then by mixed solution with containing 0.02%CaCO
3deionized water is diluted to 50%.
By after above-mentioned substratum respectively packing in 0.103MPa, 121 DEG C, autoclaving 20min.Be cooled to about 40 DEG C and make liquid nutrient medium.
2, different culture media produces spore capacity variance mensuration
To beat at the colony edge cultivating 3d with the punch tool of diameter 5mm and get bacterium block, be placed in above-mentioned 11 kinds of different liqs substratum, 5 mycelia blocks put into by every ware.Dark culturing 5d at postvaccinal culture dish is placed in 25 DEG C, outwells liquid nutrient medium, after continuing to cultivate 2d, changes a sterilized water, then cultivates 3d " Invest, Then Investigate " liquid nutrient medium miospore capsule generation with sterilized water.Each substratum repeats 3 times.Each investigation gets five pieces of 1cm by five-spot from culture dish
2the mycelia block of size, counts the sporocyst quantity that each visual field mycelia block produces under microscope 10 times of eyepieces, statistical study different liqs substratum produces the difference of sporocyst ability.
After in different liqs substratum, Phytophthora cactorum bacterium produces sporocyst in a large number, culture dish is placed in 4 DEG C of refrigerator 30-60min, take out again at being placed in 25 DEG C and cultivate, observe the ratio of Zoospore liberation in counting different liqs substratum, the difference of zoospore releasability in statistical study different liqs substratum.
Phytophthora cactorum bacterium is cultivated the ability producing Sporangia and zoospores release and supplies examination substratum (table 1) apparently higher than other in celery liquid nutrient medium.In addition, in celery liquid nutrient medium, the concentration difference of Sucus Oenanthes Javanicae also has impact to sporulation quantity and Zoospore liberation amount, celery substratum (10%) > celery substratum (25%) > celery substratum (50%).
Table 1 celery substratum and other substratum produce sporocyst capacity variance and compare
| For examination substratum | Unit surface produces sporangial amount (individual/visual field) | Zoospore liberation 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, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Claims (1)
1. one kind is that the suitable Phytophthora cactorum bacterium (Phytophthora cactorum) of raw material making produces sporangial liquid nutrient medium with celery, it is characterized in that, concrete making method is, the ratio being 1: 1 in w/v after celery defoliation cleans chopping adds deionized water and squeezes the juice in juice extractor, is used containing 0.2% (w/v) CaCO by gained juice with after four layers of filtered through gauze
3deionized water be diluted to containing Sucus Oenanthes Javanicae 10% ~ 50% (v/v), with 0.103MPa, 121 DEG C, autoclaving 20min after packing; Sterilising medium is poured in the culture dish of sterilizing and make liquid nutrient medium.
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| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
Non-Patent Citations (1)
| Title |
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| 胶孢炭疽菌及两种疫霉菌对苯酰菌胺的敏感性与β-微管蛋白氨基酸突变的相关性分析;邓维萍等;《农药学学报》;20120430;第14卷(第2期);全文 * |
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