CN1966669A - Cucumber downy mildew sporangium in vitro conservation method - Google Patents
Cucumber downy mildew sporangium in vitro conservation method Download PDFInfo
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Abstract
The invention provides a in vitro conservation method for Pseudoperonospora cubensissporangium. It includes process of: the treatment and conservation method and conditions ofPseudoperonospora cubensissporangium, the detection of sporangium germination rate and pathogenicity. The invention is to conserve thePseudoperonospora cubensissporangium in a mixed liquid containing 10% dimethyl sulfoxide and 5% skimmed milk, at -20DEG C, -70DEG C, or pretreat at -20DEG C for 24h, and conserve at -70DEG C. The best conservation method is pretreating at -20DEG C for 24h, and conserving at -70DEG C, after 12 months conservation, the sporangium germination rate is 46%, disease incidence and pathogenetic condition index are 50% and 40 respectively after inoculate to cucumber seminal leaves, high pathogenicity is preserved. The invention is fit for the long term in vitro conservation of Pseudoperonospora cubensis, and can solve the problem of serious pathogenicity descent in vitro conservation fundamentally.
Description
(1) technical field
What the present invention relates to is the long-term in-vitro conservation method of a kind of cucumber downy mildew sporangium.
(2) background technology
Cucumber downy mildew is a kind of destructive disease on the protection Viola grypoceras A. Gray.This disease is by Mastigomycotina, Oomycete, Peronosporaceae, Cuba artificial downy mildew (Pseudoperonospora cubensis (Berk.﹠amp; M.A.Curtis) Rostovzev) infect a kind of air infection diseases that causes.Up to now, not perfect to the Physiological Differentiation and the Genetic Diversity of this disease.Tracing it to its cause mainly is because Cuba artificial downy mildew is an obligate parasite, can't cultivate and preserve with artificial medium.The bacterial classification store method mainly contains at present: 1. host's live body is preserved: under the control environment condition, regularly germ is received on the new host.This is a kind of simple time-consuming method, and requires certain device, but but longer-term preservation.2. the leaf that exsomatizes is preserved: the Fu Shuyun of Agricultural University Of Shenyang (1983) has worked out the in vitro culture method of the leaf that carries disease germs, and under-5 ℃ of conditions, sick leaf can be preserved 26 days, but its sporocyst germination rate has only 3.5%.The stripped leaf preservation method of Tsai W. (1987) research in Taiwan can make the disease leaf preserve 1 month.Preserve moisture with the cotton that contains 5% sucrose liquid or finite concentration kinetin liquid Parkas hour (1989), sick leaf can be preserved 30d.3. cryogenic freezing is preserved: people such as Zhou Fengzhen report in 1987, adopt cryopreservation, and under-20 ℃ of conditions, sporocyst is preserved after 60 days and is still had virulence.Shi Yanxia (2002,2005) etc. by the research of low temperature and dry influence to germ sporocyst virulence is pointed out that low temperature (20 ℃) has reduced the sporangial virulence of Pseudoperonospora cubensis, the low temperature time is long more, sporangial virulence is low more, and low temperature prolongs the incubation period simultaneously.10 months downy mildew of the freezing preservation of excised leaf still has virulence, but virulence decline, disease index only is 1.60, and the contrast disease index has reached 96.2, and the freezing bacterial classification incubation period reaches 17d simultaneously, and the contrast incubation period is 4d.4. liquid nitrogen is preserved: Gulya reports that in 1993 the xerospore capsule of Pseudoperonospora cubensis such as Sunflower Receptacle need not cryoprotectant and can preserve in liquid nitrogen; but this method needs sporocyst is carried out dry pre-treatment; forfeiture is pathogenic easily to handle bad sporocyst; and need liquid nitrogen plant when preserving, and deposit in the liquid nitrogen throughout the year.
From above-mentioned about the store method of bacterium of downy mildew of cucumber bacterial classification as seen, up to the present also do not find ideal, simple and effective store method.Therefore the stripped long-term preservation method of germ is established in exploitation, is the basis that cucumber downy mildew is furtherd investigate.
(3) summary of the invention
The object of the present invention is to provide the stripped long-term preservation method of a kind of simple, effective bacterium of downy mildew of cucumber.
The object of the present invention is achieved like this: gather the fresh sick leaf of cucumber downy mildew, wash away foreign material and original sporocyst on the blade face, at 20 ℃～25 ℃, preserved moisture 24 hours, wait to grow after a large amount of dense fresh sporocysts, with the sterilization banister brush sporocyst is brushed 10% dimethyl sulfoxide (DMSO) and preserve in the liquid, under-20 ℃～-80 ℃ conditions, preserve then with mixing of 5% skimming milk.
Preservation of the present invention can be carried out under the following conditions:
1, under-20 ℃ of conditions, preserves.
2, under-70 ℃ of conditions, preserve.
3, under-20 ℃ of conditions, handled 24 hours earlier, under-70 ℃～-80 ℃ conditions, preserve then.
The invention has the beneficial effects as follows, the bacterium of downy mildew of cucumber sporocyst sporangial germination rate after preserving 12 months under above-mentioned preservation liquid and the three kinds of condition of different temperatures that exsomatizes is respectively 16%, 11% and 46%, and 7 days sequela rates of inoculation cucumber cotyledons are respectively 33.3%, 33.3% and 50%; Disease index is respectively 23.3,30 and 40.The germ sporocyst still keeps higher virulence.Wherein with sporocyst in advance 10% dimethyl sulfoxide (DMSO) add in the mixed solution of 5% skimming milk-20 ℃ freezing 24 hours, it is best to put into the effect of preserving under-70 ℃～-80 ℃ conditions then.Adopting said method has solved the serious problem of bacterium of downy mildew of cucumber in vitro conservation virulence decline for a long time.The sporocyst of preserving with this method can be further used for that the disease resistance of cucumber variety is identified, the research of the aspects such as heritable variation of the differentiation of bacterium of downy mildew of cucumber physiological strain, virulence.
(4), specific embodiments
For example the present invention is done more detailed description below:
Embodiment one: gather the fresh sick leaf of cucumber downy mildew, wash away foreign material and original sporocyst on the blade face, at 20 ℃～25 ℃, preserved moisture 24 hours, wait to grow after a large amount of dense fresh sporocysts, to brush weight percent concentration be 10% dimethyl sulfoxide (DMSO) and the mixing in the preservation liquid of the aqueous solution of 5% skimming milk with sporocyst with the sterilization banister brush, preserves under-20 ℃ of conditions then.
Embodiment two: gather the fresh sick leaf of cucumber downy mildew, wash away foreign material and original sporocyst on the blade face, at 20 ℃～25 ℃, preserved moisture 24 hours, wait to grow after a large amount of dense fresh sporocysts, to brush weight percent concentration be 10% dimethyl sulfoxide (DMSO) and the mixing in the preservation liquid of the aqueous solution of 5% skimming milk with sporocyst with the sterilization banister brush, preserves under-70 ℃ of conditions then.
Embodiment three: gather the fresh sick leaf of cucumber downy mildew, wash away foreign material and original sporocyst on the blade face, at 20 ℃～25 ℃, preserved moisture 24 hours, wait to grow after a large amount of dense fresh sporocysts, to brush weight percent concentration be 10% dimethyl sulfoxide (DMSO) and mixing of the aqueous solution of 5% skimming milk preserves in the liquid with sporocyst with the sterilization banister brush, and processing 24 hours under-20 ℃ of conditions is earlier preserved under-80 ℃ of conditions then.
The step of above-mentioned embodiment is:
1. bacterial classification is originated
Collection washes away foreign material and original sporocyst on the blade face from the fresh sick leaf of field generation cucumber downy mildew, at 20 ℃～25 ℃, preserves moisture 24 hours, waits to grow after a large amount of dense fresh sporocysts, is used for the preservation of germ.
2. store method and step
Preserve in the liquid with the mixing that the sterilization banister brush is brushed 10% dimethyl sulfoxide (DMSO) and 5% skimming milk with sporocyst, put into 1.5ml ep pipe.Look actual conditions then, at-20 ℃ ,-70 ℃ or under-20 ℃ of conditions, handled 24 hours earlier, under-70 ℃ different condition, preserve then.Wherein to handle 24 hours under-20 ℃ of conditions earlier, the method preservation effect of preserving under-70 ℃ of conditions is best then.
After preserving certain hour, when the disease resistance of carrying out cucumber variety is identified, during the research of the aspects such as heritable variation of the differentiation of bacterium of downy mildew of cucumber physiological strain, virulence, can at any time the sporocyst that is kept in the centrifuge tube preservation liquid be taken out from refrigerator, at first measure its sporangial germination rate and virulence, be used for above-mentioned research then.
3. preserve the required condition of bacterium of downy mildew of cucumber
Gather the sick leaf of cucumber downy mildew and carry out above-mentioned processing at the cucumber downy mildew occurance peak.
-20 ℃ and-70 ℃～-80 ℃ refrigerators.
The bacterium of downy mildew of cucumber sporocyst is sprouted and is inoculated pathogenic evaluation condition artificial seedling stage.
4. technical essential of the present invention
One of technical essential of the present invention is that stripped sporocyst is kept in the mixing preservation liquid of 10% dimethyl sulfoxide (DMSO) and 5% skimming milk.Dimethyl sulfoxide (DMSO) has another name called methyl sulfoxide, sulfinyl bis methane, is called for short DMSO.Be a kind of important fine chemical material, have high polarity, high-hygroscopicity, high boiling point and combustibility, toxicity is extremely low, and Heat stability is good is a kind of intensive polar solvent; The freezing point of dimethyl sulfoxide (DMSO) is 18.45 ℃, and boiling point is 189.0 ℃, has the dual function of high-temperature solvent and low-temperature solvent concurrently.Medically can be used for frostproofer that marrow, blood, organ hypothermia preserves etc.This research promptly according to these characteristics of dimethyl sulfoxide (DMSO), is used for the preservation of Pseudoperonospora cubensis.In preserving liquid, add a certain amount of skimming milk simultaneously, offer pathogenic bacteria, prolong preservation period with certain nutrition.
Two of technical essential of the present invention is that the sporocyst of preserving in the liquid is preserved at low temperatures.Can be according to actual conditions at-20 ℃ ,-70 ℃～-80 ℃ or earlier-20 ℃ of pre-treatment 24 hours, then-70 ℃ of preservations.Wherein with pre-treatment under-20 ℃ of conditions 24 hours, the sporocyst virulence of preserving under-70 ℃ of conditions was the strongest then, and preservation effect is best.
5. sporangial virulence measuring method behind the preservation certain hour
(1) mensuration of sporocyst germination rate
On the slide glass of sterilization, splash into the sporocyst solution of preserving under the above different condition, put into the 20 ℃ of heat insulating culture of sterile petri dish that are lined with wet filter paper, begin to observe, observe 10 visuals field at every turn from cultivating back 1 hour.Each is handled 3 times and repeats.Record sporocyst germination rate.With the sporocyst on the new scab of sick leaf then is contrast.
(2) virulence measuring method
Adopt cotyledon artificial inoculation method to measure sporangial pathogenic.The host selects the susceptible variety Chang Chun Mi Ci for use.To be seeded in after its seed disinfection in the sterilization soil, 3 repetitions are established in every processing, amount to 30 alms bowls.Going bail at cotyledon period is stored in sporangia suspension under the above-mentioned different condition, and concentration is that 2000 sporocyst/ml carry out the drop inoculation, and inoculum size is 0.04ml.Inoculation is placed on and prevents in the humidistat that pollute in other bacterium sources in the air.And to keep temperature daytime be 20 ℃～25 ℃, and at 10 ℃～15 ℃ of nights, relative humidity 90%～100% is inoculated back 7 days investigation sickness rate and disease index.State of an illness grade scale is as follows:
0 grade: no illness
1 grade: the inoculation point has small scab, and its diameter is less than 0.5cm
2 grades: scab diameter 0.5～1.3cm
3 grades: inoculation point yellow area accounts for below 1/3 of whole area, and necrotic plaque accounts for below 1/3
4 grades: the necrotic plaque area accounts for 1/3～2/3 of whole area
5 grades: the necrotic plaque area account for whole area more than 2/3 or complete stool withered
Calculate disease index, formula is:
Claims (5)
1, a kind of cucumber downy mildew sporangium in vitro conservation method, it is characterized in that: gather the fresh sick leaf of cucumber downy mildew, wash away foreign material and original sporocyst on the blade face, at 20 ℃～25 ℃, preserved moisture 24 hours, wait to grow after a large amount of dense fresh sporocysts, with the sterilization banister brush sporocyst is brushed 10% dimethyl sulfoxide (DMSO) and preserve in the liquid, under-20 ℃～-80 ℃ conditions, preserve then with mixing of 5% skimming milk.
2, cucumber downy mildew sporangium in vitro conservation method according to claim 1 is characterized in that: preserve under-20 ℃ of conditions.
3, cucumber downy mildew sporangium in vitro conservation method according to claim 1 is characterized in that: preserve under-70 ℃ of conditions.
4, cucumber downy mildew sporangium in vitro conservation method according to claim 1 is characterized in that: handled 24 hours under-20 ℃ of conditions earlier, preserve under-70 ℃～-80 ℃ conditions then.
5, cucumber downy mildew sporangium in vitro conservation method according to claim 4 is characterized in that: handled 24 hours under-20 ℃ of conditions earlier, preserve under-70 ℃ of conditions then.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102972302A (en) * | 2012-12-27 | 2013-03-20 | 新疆农业科学院哈密瓜研究中心 | Method for preserving melon powdery mildew fungus |
| CN106755250A (en) * | 2016-12-27 | 2017-05-31 | 山东金晶生物技术有限公司 | A kind of preservation of haematococcus pluvialis green cell and the large-scale method for producing of astaxanthin induction |
| CN109328683A (en) * | 2018-09-30 | 2019-02-15 | 山西省农业科学院植物保护研究所 | A method of quinoa downy mildew disease resistance is identified using cutting propagation |
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2006
- 2006-11-24 CN CNB2006101510558A patent/CN100489084C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102972302A (en) * | 2012-12-27 | 2013-03-20 | 新疆农业科学院哈密瓜研究中心 | Method for preserving melon powdery mildew fungus |
| CN106755250A (en) * | 2016-12-27 | 2017-05-31 | 山东金晶生物技术有限公司 | A kind of preservation of haematococcus pluvialis green cell and the large-scale method for producing of astaxanthin induction |
| CN106755250B (en) * | 2016-12-27 | 2020-06-02 | 山东金晶生物技术有限公司 | Haematococcus pluvialis green cell preservation and astaxanthin-induced large-scale production method |
| CN109328683A (en) * | 2018-09-30 | 2019-02-15 | 山西省农业科学院植物保护研究所 | A method of quinoa downy mildew disease resistance is identified using cutting propagation |
| CN109328683B (en) * | 2018-09-30 | 2020-09-18 | 山西农业大学 | Method for identifying downy mildew disease resistance of quinoa by utilizing cutting propagation |
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| CN100489084C (en) | 2009-05-20 |
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