CN1693447A - Process for preserving bacterial strain of cucumber downy mildew bacteria - Google Patents
Process for preserving bacterial strain of cucumber downy mildew bacteria Download PDFInfo
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- CN1693447A CN1693447A CN 200510070978 CN200510070978A CN1693447A CN 1693447 A CN1693447 A CN 1693447A CN 200510070978 CN200510070978 CN 200510070978 CN 200510070978 A CN200510070978 A CN 200510070978A CN 1693447 A CN1693447 A CN 1693447A
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Abstract
A method for storing the downy mildew strain of cucumber includes such steps as disinfecting the leaf of cucumber under aseptic condition, preparing leaf discs, dripping the sporange suspension of said downy mildew strain on the back of said leaf discs, culturing until black midew layer is generated, and storing it at -30- -80 deg.C in sealed mode.
Description
Technical field
The present invention relates to the store method of a kind of obligate parasite in the plant protection field, particularly relate to a kind of store method of bacterial strain of cucumber downy mildew bacteria.
Background technology
Cucumber is the main varieties of plant of China's vegetables producing region open country and the cultivation of protection ground, can plant the whole year in most areas.Oidium is that cucumber produce to go up one of most important disease, and all there is generation in the area of all cultivated cucumbers, and it is the heaviest to be injured with northern area especially in China, and outdoor cropping is the heaviest with spring stubble cucumber morbidity, and spring is cultivated then on protection ground or fall ill heavier winter.This disease is mainly propagated by air-flow, has to infect characteristics frequent, that the incubation period is short, and popular harm is serious, can in several days, cause plant all dead, be commonly called as " horse race is done ", also because of after its morbidity, the mustiness thing of a large amount of puces appears in the cucumber blade back, and is referred to as " black wool disease ".
The pathogenic bacteria of cucumber downy mildew is Cuba artificial downy mildew (Pseudoperonospora cubensis (Berk.EtCert.) Rostr.), this bacterium is a kind of obligate parasite, mycelia mainly expands in the host cell gap after its spore germination, can not on artificial medium, grow, so this bacterial strain is difficult to carry out secular preservation under isolated condition.Many research institutions both domestic and external and farmingization enterprise are now just stepping up to develop the new type bactericide that these pathogenic bacterium is had effective prevention effect.Need use these a large amount of live body pathogenic bacterium when the different sterilant of developing using carry out toxicity test to bacterium of downy mildew of cucumber, therefore press for a kind of long-term preservation method of bacterial strain of cucumber downy mildew bacteria.
Summary of the invention
The longer store method that the purpose of this invention is to provide a kind of bacterial strain of cucumber downy mildew bacteria.
The store method of bacterial strain of cucumber downy mildew bacteria provided by the present invention, be under aseptic condition, make the leaf dish with the healthy excised leaf on disinfectant cucumber plant 3-6 leaf position, each leaf dish back side is dripped the sporangia suspension of bacterium of downy mildew of cucumber, then it is preserved moisture under 17-20 ℃ of dark condition and cultivate 20-28h, again in the cultivation of preserving moisture greater than 80%, under the 10-14h alternation of light and darkness condition of 17-20 ℃, humidity RH, after treating that the leaf dish back side produces the mould layer of black, place-30 ℃ to-80 ℃ airtight preservations down.
In the aforesaid method, described healthy excised leaf is preferably the blade on cucumber plant the 4th leaf position; The leaf dish is of a size of Φ 1.2-2.0cm, is preferably Φ 1.5cm; The concentration of the sporangia suspension of bacterium of downy mildew of cucumber is 1 * 10
4-1 * 10
5Individual sporocyst/mL is preferably 1.0 * 10
4Individual sporocyst/mL; The volume of the sporangia suspension that each leaf dish back side drips is 10 μ L; The culture condition that the back side drips the leaf dish of the sporangia suspension that bacterium of downy mildew of cucumber is arranged is preferably to preserve moisture under 19 ℃ dark condition earlier cultivates 24h, again in the cultivation of preserving moisture greater than 80%, under the 12h alternation of light and darkness condition of 19 ℃, humidity RH; Storage temperature is preferably-80 ℃.
The preparation method of described sporangia suspension is; The sick leaf that leaf back has the mould layer of black is gathered in the field, indoor impurity on the sick leaf and aging sporocyst are cleaned after, at 19 ℃ of dark down sporocysts of inducing generation new of cultivating, again newborn sporocyst is inoculated into the stripped cucumber leaves back side of aseptic health, placing preserves moisture under the 17-20 ℃ of dark condition cultivates 20-28h, again in 17-20 ℃, humidity RH is greater than 80%, the cultivation of preserving moisture under the 10-14h alternation of light and darkness condition, after treating that vacuum side of blade produces the mould layer of black, the single sporangiophore of picking carries out the purifying of bacterial strain, and with single sporangiophore and the sporocyst of giving birth to be transferred to cultivations of under 17-20 ℃, preserving moisture of the excised leaf back side on the healthy cucumber 3-5 leaf position, the morbidity afterwards sporocyst on the wash-out scab is made into sporangia suspension.
In the preparation method of above-mentioned sporangia suspension, newborn sporangial inoculation method is a sessile drop method, and the concentration of used newborn sporangia suspension is preferably 1.0 * 10 when inoculating with sessile drop method
4Individual sporocyst/mL, the inoculation volume is 10 μ L; Back side inoculation has the culture condition of the sporangial cucumber leaves of bacterium of downy mildew of cucumber to be preferably to preserve moisture under 19 ℃ dark condition earlier cultivates 24h, cultivates greater than 80%, under the 12h alternation of light and darkness condition in 19 ℃, humidity RH again.
In actual applications, the bacterial strain of cucumber downy mildew bacteria that aforesaid method is preserved can be put into ice chest and make its slow intensification, ftracture in order to avoid sporocyst is shock heating, the wash-out sporocyst is made sporangia suspension and can be used then.
The characteristics that the present invention can be survived at low temperatures according to bacterium of downy mildew of cucumber provide easy, the permanent method of preserving bacterial strain of cucumber downy mildew bacteria under a kind of deepfreeze condition.Can make bacterial strain of cucumber downy mildew bacteria in 60 days, all keep greater activity, the toxicity test method of routines such as available leaf dish floating method is carried out toxicity test to it, measurement result shows that the virulence of bacterial strain in 60d of preserving with this method is all consistent with blank, no significant difference, can be used for the toxicity test of sterilant, for the control of studying cucumber downy mildew provides very big convenience to bacterium of downy mildew of cucumber.The present invention has higher actual application value.
Description of drawings
Fig. 1 has the sick leaf of the mould layer of obvious brown for the leaf back of gathering from the field disease plant
Fig. 2 is for inducing the new sporocyst of generation on sick leaf
Fig. 3 has the stripped cucumber leaves of health of newborn sporangia suspension for inoculation
The blade of the cucumber plant that Fig. 4 exsomatizes for the health that inoculation is had newborn sporangia suspension is secretly cultivated the back and is cultivated greater than 80%, under the condition of 12h alternation of light and darkness in 19 ℃, humidity RH
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The test plant material:
Susceptible cucumber variety Chang Chun Mi Ci is cultivated the 15cm in Φ, in the pot for growing seedlings of high 12cm, place glasshouse to cultivate, treat the long blade of plant, be used for the cultivation of bacterium of downy mildew of cucumber and the cryogenic freezing preservation of bacterial strain to 6 leaf after dates collection healthy plant the 4th leaf position.
The separation and Culture of embodiment 1, bacterium of downy mildew of cucumber and preservation
1, the separation and Culture of collection of cucumber downy mildew sample and germ
Gather the bacterium of downy mildew of cucumber sample from the cucumber cultivation ground that flumorph and similar medicament dimethomorph were not used in Dong Gaocun Cui village Technology Park, Pinggu, Wang Huacun western food ground, Huairou, Kong Huaying village, Yongning, Yanqing town, Hao Zhuan apple garden, south, Changping and the areas such as China Agricultural University's Changping experiment centre, beans shop, Fangshan village, northwest, Haidian District Wang Cun and Hebei mountains in a range are flat, Inner Mongol of Beijing suburb, be divided into from obtaining 77 strain sensitive strains, separate obtaining 6 strains have different resistance levels to metaxanin drug-fast strain simultaneously from Beijing suburb; Obtain 6 strain nitrile Azoxystrobin drug-fast strains from the booth separation of carrying out 2 years nitrile Azoxystrobin field control effectiveness tests.Concrete grammar is: will have the sick leaf (Fig. 1) of the mould layer of obvious brown with distilled water remaining impurities on the blade and aging sporocyst to be cleaned from the leaf back that the field disease plant is gathered, place dark culturing 24h under 19 ℃ the condition then, induce to produce new sporocyst (Fig. 2).Sporocyst wash-out with 4 ℃ of sterilized waters will newly produce is made into every milliliter and contains 1 * 10
4Individual sporangial suspension, be inoculated into the vacuum side of blade of the cucumber plant that exsomatizes with the health behind the aseptic water washing with sessile drop method, every 10 μ L, be placed on again in the Φ 150mm culture dish of preserving moisture and cultivate 24h (Fig. 3) down in 19 ℃ of dark conditions with thieving paper, to guarantee the intrusion of pathogenic bacteria, cultivate (Fig. 4) in 19 ℃, humidity RH greater than 80%, under the condition of 12h alternation of light and darkness then, blade can be observed irregular water soaking mode scab after meeting bacterium 3d, and the 5d rear blade back side produces the mould layer of black.The single sporangiophore of picking carries out the purifying of bacterial strain under anatomical lens, and with single sporangiophore of picking and the sporocyst of giving birth to transfer to the cultivation of preserving moisture of the healthy stripped cucumber leaves back side, pathogenic bacteria can preserve 10-15d on the cucumber excised leaf.Can continue to be transferred to succeeding transfer culture on the fresh healthy cucumber excised leaf with the sporocyst on 4 ℃ of sterilized water wash-out scabs.
2, the deepfreeze of bacterium of downy mildew of cucumber is preserved
Select the healthy excised leaf of cucumber plant the 4th leaf position, after carrying out surface sterilization and handling, produce the Folium Cucumidis sativi dish of Φ 1.5cm with punch tool, it is 1 * 10 that each leaf dish back side drips the concentration of separating the sporocyst preparation of the bacterium of downy mildew of cucumber that obtains with aforesaid method
4The sporangia suspension 10 μ L of individual sporocyst/mL, cultivate according to the described method of step 1 then, after treating that the leaf dish back side produces the mould layer of black, in its 10mL tool plug centrifuge tube of carefully packing into, place 4 ℃ respectively,-20 ℃,-30 ℃, to preserve down for-70 ℃ and-80 ℃, each temperature repeats to place 10 pipes, get 1 pipe every 10d and put into ice chest and slowly heat up, ftracture in order to avoid sporocyst is shock heating.Wash-out sporocyst then, make the sporangia suspension that concentration is 1 * 104 sporocyst/mL, it is seeded on the Folium Cucumidis sativi dish, the cultivation of preserving moisture, lesion area is measured in the morbidity back, relatively the variation of the down different shelf time germ virulencies of each storage temperature, bacterium of downy mildew of cucumber 4 ℃ of following shelf times less than 10d, can preserve 20d under-20 ℃, can preserve 40d at least under-30 ℃, can preserve at least more than the 60d under-70 ℃ and-80 ℃.But preserve under-20 ℃ and-30 ℃ of conditions, grow with time, the bacterial classification virulence weakens; And under-70 ℃ and-80 ℃ of conditions, the bacterial classification virulence is consistent with blank in the 60d, does not have significant difference.When shelf time during greater than 30d, the virulence of bacterial classification is better than the bacterial classification that other condition is handled under-80 ℃ of conditions.
Table 1 bacterium of downy mildew of cucumber refrigerates the variation of its virulence behind the different time under cold condition
| Shelf time (d) | Lesion area (mm 2) | ||||
| ????4℃ | ????-20℃ | ??-30℃ | ????-70℃ | ????-80℃ | |
| ????10 ????20 ????30 ????40 ????50 ????60 | ??0.00?a ????- ????- ????- ????- | ??143.08?a ???79.33?b ????0.00?c ????0.00?c ??????- ??????- | 110.05?a ?89.09?b ?57.27?c ?36.67?d ??0.00?e ????- | ?116.38?a ??97.09?a ?100.36?a ?101.22?a ?111.75?a ?110.73?a | ?113.22?b ?117.61?ab ?101.56?b ?140.22?a ?144.00?a ?111.79?b |
Annotate: a=0.05;-do not do inoculation experiments
Claims (9)
1, a kind of store method of bacterial strain of cucumber downy mildew bacteria, be under aseptic condition, the leaf dish made from the healthy excised leaf on disinfectant cucumber plant 3-6 leaf position, each leaf dish back side is dripped the sporangia suspension of bacterium of downy mildew of cucumber, then it is preserved moisture under 17-20 ℃ of dark condition and cultivate 20-28h, the cultivation of under 17-20 ℃, humidity RH＞80%, alternation of light and darkness condition, preserving moisture again, treat that the leaf dish back side produces the mould layer of black after, place-30 ℃ to-80 ℃ times airtight preservations.
2, store method according to claim 1 is characterized in that: described healthy excised leaf is the blade on cucumber plant the 4th leaf position.
3, store method according to claim 1 is characterized in that: described leaf dish is of a size of φ 1.2-2.0cm; The concentration of the sporangia suspension of bacterium of downy mildew of cucumber is 1 * 10
4-1 * 10
5Individual sporocyst/mL; The volume of the sporangia suspension that each leaf dish back side drips is 10 μ L.
4, store method according to claim 3 is characterized in that: described leaf dish is of a size of φ 1.5cm; The concentration of the sporangia suspension of bacterium of downy mildew of cucumber is 1.0 * 10
4Individual sporocyst/mL; The volume of the sporangia suspension that each leaf dish back side drips is 10 μ L.
5, according to claim 1 or 2 or 3 or 4 described store methods, it is characterized in that: the described back side drips the culture condition of the leaf dish of the sporangia suspension that bacterium of downy mildew of cucumber is arranged and cultivate 24h for preserving moisture earlier under 19 ℃ dark condition, again in the cultivation of preserving moisture greater than 80%, under the alternation of light and darkness condition of 19 ℃, humidity RH.
6, according to claim 1 or 2 or 3 or 4 described store methods, it is characterized in that: described storage temperature is-80 ℃.
7, according to claim 1 or 2 or 3 or 4 described store methods, it is characterized in that: the preparation method of described sporangia suspension is: after the impurity on the sick leaf and aging sporocyst are cleaned, at 19 ℃ of dark down sporocysts of inducing generation new of cultivating, again newborn sporocyst is inoculated into the stripped cucumber leaves back side of aseptic health, placing preserves moisture under the 17-20 ℃ of dark condition cultivates 20-28h, again in 17-20 ℃, humidity RH＞80%, the cultivation of preserving moisture under the alternation of light and darkness condition, after treating that vacuum side of blade produces the mould layer of black, the single sporangiophore of picking carries out the purifying of bacterial strain, and with single sporangiophore and the sporocyst of giving birth to be transferred to cultivations of under 17-20 ℃, preserving moisture of the excised leaf back side on the healthy cucumber 3-6 leaf position, the morbidity afterwards sporocyst on the wash-out scab is made into sporangia suspension.
8, store method according to claim 7 is characterized in that: the sporangial inoculation method of described new life is a sessile drop method, and the concentration of used newborn sporangia suspension is 1.0 * 10 during inoculation
4Individual sporocyst/mL, the inoculation volume is 10 μ L.
9, store method according to claim 7, it is characterized in that: the inoculation of the described back side has the culture condition of the sporangial cucumber leaves of bacterium of downy mildew of cucumber to cultivate 24h for preserving moisture earlier under 19 ℃ dark condition, the cultivation of preserving moisture under 19 ℃, RH＞80%, alternation of light and darkness condition again.
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| CN1269140A (en) * | 1999-03-29 | 2000-10-11 | 黄志桂 | Composition for preventing and controlling plant diseases |
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