CN108315260A - A kind of in-vitro conservation method of melon powdery mildew germ - Google Patents
A kind of in-vitro conservation method of melon powdery mildew germ Download PDFInfo
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Abstract
The invention belongs to biopathogen bacterium Techniques of preserving fields, and in particular to a kind of in-vitro conservation method of melon powdery mildew germ.This approach includes the following steps：Step 1：Culture medium is prepared, is then added to culture medium in the culture dish after high pressure steam sterilization；Step 2：After culture medium solidification, 1~2 layer of blotting paper is put on culture medium.Step 3：The seed for feeling powdery mildew melon variety is carried out water planting to take the cotyledon of seedling after seedling grows the 1st true leaf, then lie in the cotyledon after disinfection on blotting paper；Step 4：Powdery mildew germ is taken, cotyledon is inoculated with.Step 5：Culture dish lid is covered after inoculation, and culture dish is placed in 20 DEG C of temperature, 2000 lux of illuminance, photoperiod（Day/night）It is cultivated under 12 hours/12 hours environment；Step 6：After 35~42 days, transfer to the epicotyledonary powdery mildew germ.This method operation is very easy, and at low cost, the germ holding time is longer.
Description
Technical field
The invention belongs to biopathogen bacterium Techniques of preserving fields, and in particular to a kind of Plantlet in vitro of melon powdery mildew germ
Method.
Background technology
Muskmelon is Curcurbitaceae（Cucurbitaceae）Cucumis（CucumisL.）Annual sprawling herbs plant.China
There are long melon wilt history, and maximum muskmelon production and consumption big country in the world.It is counted according to FAO, sweet tea in 2014
Melon cultivated area reaches 43.89 ten thousand hm2, yield accounted for the 50.04% of Gross World Product.However, with protecting field sweet tea in recent years
The expansion of melon cultivated area, muskmelon disease are on the rise.Powdery mildew is one of the important disease in the production of protecting field muskmelon, mainly
The plant leaf of Muskmelon Seedlings phase and Adult plant is infected, can also be infected on serious period stem and fruit.In suitable environment
Under the conditions of, powdery mildew development is very fast, and several days time can spread to entire greenhouse or melon patch, causes yellow leaf, dries up,
The photosynthesis of plant is seriously affected, and then influences the yield and quality of melon fruit.
In view of the serious harm of melon powdery mildew, have become a hot spot of researcher research.However, muskmelon
Genus erysiphe in obligate parasite, no matter its mycelium or the conidium in imperfect stage, after leaving live body host, life
Holding time for power is all very short, therefore Powdery Mildew must carry out live body preservation.Current research personnel are carrying out melon powdery mildew
In research, in order to ensure that the anniversary of powdery mildew germ preserves, it usually needs constantly culture disease plant seedling passes through spore suspension
Liquid spray inoculation, and germ of constantly transferring.Although also having been reported that when plant grows to 5 true leaves, clip shoot tip plant is to filling
In the triangular flask of benzimidazole-mannite agar sugar culture-medium, then it is inoculated with and preserves on stem apex.Due to that must wait until that plant is long
To can just take stem apex when 5~6 leaf, time-consuming, cost is higher, and be easy to cause pollution during Shoot Tip Culture.Traditional
Spore suspension spray inoculation store method, it is necessary to nursery is carried out, is inoculated with until plant grows to 3~4 true leaves, and
Plant morbidity rear blade turns yellow, is withered quickly, and not only seedling cost is high, and the holding time is also short.
Invention content
The purpose of the present invention is to overcome the deficiency in the prior art, provides a kind of Plantlet in vitro side of melon powdery mildew germ
Method, this method are inoculated with by Cotyledon Segments, substantially reduce the incubation time of Muskmelon Seedlings early period, easy to operate, at low cost；Together
When sprouted due to seed after most of nutrition be stored in cotyledon, compared with true leaf, the germ holding time is inoculated with cotyledon
It is longer；In addition, there is being isolated for blotting paper between cotyledon and culture medium, the probability of pollution is reduced.
To achieve the above object, the present invention uses following technical scheme：
A kind of in-vitro conservation method of melon powdery mildew germ, includes the following steps：
Step 1：Prepare culture medium：In every 1000 milliliters of sterile waters be added 8 grams of agar, 6.8 grams of sucrose, 18.2 grams of mannitol and
The Natamycin solution of 400 microlitres of mass fractions 25%；Then culture medium is added in the culture dish after high pressure steam sterilization.
Step 2：After culture medium solidification, the blotting paper after 1~2 layer of high pressure steam sterilization is put on culture medium.
Step 3：The seed for feeling powdery mildew melon variety is subjected to water planting, after seedling grows the 1st true leaf, in ultra-clean work
The cotyledon for making to take seedling on platform, with aseptic water washing 3 times after cotyledon is impregnated 3 seconds in the alcohol of percentage by volume 75%, so
Cotyledon is lain in culture dish afterwards, the lower surface of the cotyledon is downward.
The water planting includes the following steps：
The seed for feeling powdery mildew melon variety is soaked seed 3~4 hours in 55 DEG C of warm water, is then placed in the training for being covered with wetting filter paper
It supports in ware, and covers culture dish lid；
Culture dish is placed under the dark surrounds of 28~30 DEG C of temperature to cultivate 18~24 hours and carries out vernalization；
After germination, culture dish is placed on 25 DEG C of temperature, 6000 lux of illuminance, 12 hours/12 hours photoperiod（Light
According to/dark）It is cultivated under environment；
When seedling height is close to culture dish height, opens culture dish lid and cultivated；
Growth of seedling situation is observed, supplements 1/2 Hogland nutrient solutions into culture dish in time, keeps filter paper wetting until seedling
Rough leaf is fully deployed.
Step 4：Melon powdery mildew disease plant is chosen in muskmelon producing region, first with ear washing bulb by melon powdery mildew disease plant
The old spore on the powdery mildew scab surface on blade is blown off, is taken with oese within second day fresh in diameter 3mm powdery mildew scabs
Spore is inoculated on cotyledon described in step 3.
Step 5：Culture dish lid is covered after inoculation, and culture dish is placed on 20 DEG C of temperature, 2000 lux of illuminance, light
12 hours/12 hours period（Illumination/dark）It is cultivated under environment.
Step 6：After 35~42 days, transfer to the epicotyledonary powdery mildew germ.
Further, there is circular hole, a diameter of 4~6mm of the circular hole, under the petiole of the cotyledon on the blotting paper
End is located in circular hole, and is contacted with culture medium.
Further, the culture medium thickness in the culture dish is 2~4mm.
Further, the sense powdery mildew melon variety is the melon variety of middle sense powdery mildew or high sense powdery mildew.
Compared with prior art, the beneficial effects of the present invention are：
1, the present invention preserves it by being inoculated with powdery mildew germ on muskmelon Excised cotyledons, is opened up completely in two panels cotyledon
It opens, inoculation powdery mildew germ can be carried out after rough leaf grows, compared with traditional inoculation plant using spore suspension,
Without waiting until that plant grows up, greatly improve the efficiency；And it due to being stored with nutrition more more than true leaf in cotyledon, thus preserves
Time is also longer.
2, Natamycin is added in the culture medium used in the present invention, can effectively prevents culture medium by other bacteriums and puts
The pollution of line bacterium, has in addition been capped blotting paper on culture medium, avoids coming into contact in a large area for cotyledon and culture medium, further drops
The probability of low pollution.
3, in prioritization scheme of the present invention, there is circular hole, the petiole lower end of cotyledon to be located on blotting paper in circular hole, son can be made
Leaf can directly be absorbed into nutrition, and in addition the blade-section of cotyledon is located on blotting paper, is not contacted with culture medium, pollution probability compared with
It is small.
4, the method for the present invention carries out powdery mildew germ preservation using culture dish, and culture medium dosage is few, has saved cost.
5, the method for the present invention is mainly used for powdery mildew germ short-term preservation, meets the need using powdery mildew germ in the short time
It asks, cultivation cycle is short, and operation is very easy, at low cost.
Description of the drawings
Fig. 1 is melon powdery mildew germ Plantlet in vitro figure in the embodiment of the present invention 2.
Specific implementation mode
Following embodiment is not used to limit protection scope of the present invention for illustrating the present invention.Unless otherwise specified, real
Apply the conventional means that technological means used in example is well known to those skilled in the art.
Embodiment 1
A kind of in-vitro conservation method of melon powdery mildew germ, includes the following steps：
Step 1：Prepare culture medium：Into 1000 milliliters of sterile waters be added 8 grams of agar, 6.8 grams of sucrose, 18.2 grams of mannitol and
The Natamycin solution of 400 microlitres of mass fractions 25%；Then culture medium is added in the culture dish after high pressure steam sterilization.
Step 2：After culture medium solidification, the blotting paper after 1 layer of high pressure steam sterilization is put on culture medium.
Step 3：The seed for feeling powdery mildew melon variety is subjected to water planting, after seedling grows the 1st true leaf, in ultra-clean work
The cotyledon for making to take seedling on platform, with aseptic water washing 3 times after cotyledon is impregnated 3 seconds in the alcohol of percentage by volume 75%, so
Cotyledon is lain on the blotting paper in culture dish afterwards, the lower surface of the cotyledon is downward；The sense powdery mildew melon variety is
The melon variety of middle sense powdery mildew.
The water planting includes the following steps：
The seed for feeling powdery mildew melon variety is soaked seed 3 hours in 55 DEG C of warm water, is then placed in the culture for being covered with wetting filter paper
In ware, and cover culture dish lid；
Culture dish is placed under the dark surrounds of 28 DEG C of temperature to cultivate 24 hours and carries out vernalization；
After germination, culture dish is placed on 25 DEG C of temperature, 6000 lux of illuminance, 12 hours/12 hours photoperiod（Light
According to/dark）It is cultivated under environment；
When seedling height is close to culture dish height, opens culture dish lid and cultivated；
Growth of seedling situation is observed, supplements 1/2Hogland nutrient solutions into culture dish in time, keeps filter paper wetting until seedling
Rough leaf is fully deployed.
Step 4：Melon powdery mildew disease plant is chosen in muskmelon producing region, first with ear washing bulb by melon powdery mildew disease plant
The old spore on the powdery mildew scab surface on blade is blown off, is taken with oese within second day fresh in diameter 3mm powdery mildew scabs
Spore is inoculated on cotyledon described in step 3.
Step 5：Culture dish lid is covered after inoculation, and culture dish is placed on 20 DEG C of temperature, 2000 lux of illuminance, light
12 hours/12 hours period（Illumination/dark）It is cultivated under environment.
Step 6：After 35 days, transfer to the epicotyledonary powdery mildew germ.
Embodiment 2
A kind of in-vitro conservation method of melon powdery mildew germ, includes the following steps：
Step 1：Prepare culture medium：8 grams of agar, 6.8 grams of sucrose and 18.2 grams of mannitol are added into 1000 milliliters of sterile waters, so
After be heated to agar thawing, then culture medium solidify before be added 400 microlitres of mass fractions 25% Natamycin solution；Then
Culture medium is added in the culture dish after high pressure steam sterilization, the culture medium thickness in the culture dish is 4mm.
Step 2：After culture medium solidification, the blotting paper after 1 layer of high pressure steam sterilization is put on culture medium, on the blotting paper
With circular hole, a diameter of 6mm of the circular hole.
Step 3：The seed for feeling powdery mildew melon variety is subjected to water planting, after seedling grows the 1st true leaf, in ultra-clean work
The cotyledon for making to take seedling on platform, with aseptic water washing 3 times after cotyledon is impregnated 3 seconds in the alcohol of volume fraction 75%, then
Cotyledon is lain on the blotting paper in culture dish, the lower surface of the cotyledon is downward, and the petiole lower end of cotyledon is located in step 2
In the circular hole, and contacted with culture medium；The sense powdery mildew melon variety is the melon variety of high sense powdery mildew.
The water planting includes the following steps：
The seed for feeling powdery mildew melon variety is soaked seed 4 hours in 55 DEG C of warm water, is then placed in the culture for being covered with wetting filter paper
In ware, and cover culture dish lid；
Culture dish is placed under the dark surrounds of 30 DEG C of temperature to cultivate 18 hours and carries out vernalization；
After germination, culture dish is placed on 25 DEG C of temperature, 6000 lux of illuminance, 12 hours/12 hours photoperiod（Light
According to/dark）It is cultivated under environment；
When seedling height is close to culture dish height, opens culture dish lid and cultivated；
Growth of seedling situation is observed, supplements 1/2Hogland nutrient solutions into culture dish in time, keeps filter paper wetting until seedling
Rough leaf is fully deployed.
Step 4：Melon powdery mildew disease plant is chosen in muskmelon producing region, first with ear washing bulb by melon powdery mildew disease plant
The old spore on the powdery mildew scab surface on blade is blown off, is taken with oese within second day fresh in diameter 3mm powdery mildew scabs
Spore is inoculated on cotyledon described in step 3.
Step 5：Culture dish lid is covered after inoculation, and culture dish is placed on 20 DEG C of temperature, 2000 lux of illuminance, light
12 hours/12 hours period（Illumination/dark）It is cultivated under environment.
Step 6：After 42 days, transfer to the epicotyledonary powdery mildew germ.
Embodiment 3
A kind of in-vitro conservation method of melon powdery mildew germ, includes the following steps：
Step 1：Prepare culture medium：16 grams of agar, 13.6 grams of sucrose and 36.4 grams of mannitol are added into 2000 milliliters of sterile waters,
It is then heated to agar thawing, the Natamycin solution of 800 microlitres of mass fractions 25% is then added before culture medium solidifies；So
Culture medium is added to afterwards in the culture dish after high pressure steam sterilization, the culture medium thickness in the culture dish is 3mm.
Step 2：After culture medium solidification, the blotting paper after 1 layer of high pressure steam sterilization is put on culture medium, on the blotting paper
With circular hole, a diameter of 5mm of the circular hole.
Step 3：The seed for feeling powdery mildew melon variety is subjected to water planting, after seedling grows the 1st true leaf, in ultra-clean work
The cotyledon for making to take seedling on platform, with aseptic water washing 3 times after cotyledon is impregnated 3 seconds in the alcohol of volume fraction 75%, then
Cotyledon is lain on the blotting paper in culture dish, the lower surface of the cotyledon is downward, and the petiole lower end of cotyledon is located in step 2
In the circular hole, and contacted with culture medium；The sense powdery mildew melon variety is the melon variety of middle sense powdery mildew；The water planting
Step is the same as embodiment 2.
Step 4：Melon powdery mildew disease plant is chosen in muskmelon producing region, first with ear washing bulb by melon powdery mildew disease plant
The old spore on the powdery mildew scab surface on blade is blown off, is taken with oese within second day fresh in diameter 3mm powdery mildew scabs
Spore is inoculated on cotyledon described in step 3.
Step 5：Culture dish lid is covered after inoculation, and culture dish is placed on 20 DEG C of temperature, 2000 lux of illuminance, light
12 hours/12 hours period（Illumination/dark）It is cultivated under environment.
Step 6：After 40 days, transfer to the epicotyledonary powdery mildew germ.
Embodiment 4
A kind of in-vitro conservation method of melon powdery mildew germ, includes the following steps：
Step 1：Prepare culture medium：8 grams of agar, 6.8 grams of sucrose and 18.2 grams of mannitol are added into 1000 milliliters of sterile waters, so
After be heated to agar thawing, then culture medium solidify before be added 400 microlitres of mass fractions 25% Natamycin solution；Then
Culture medium is added in the culture dish after high pressure steam sterilization, the culture medium thickness in the culture dish is 2mm.
Step 2：After culture medium solidification, the blotting paper after 2 layers of high pressure steam sterilization is put on culture medium, on the blotting paper
With circular hole, a diameter of 4mm of the circular hole.
Step 3：The seed for feeling powdery mildew melon variety is subjected to water planting, after seedling grows the 1st true leaf, in ultra-clean work
The cotyledon for making to take seedling on platform, with aseptic water washing 3 times after cotyledon is impregnated 3 seconds in the alcohol of volume fraction 75%, then
Cotyledon is lain on the blotting paper in culture dish, the lower surface of the cotyledon is downward, and the petiole lower end of cotyledon is located in step 2
In the circular hole, and contacted with culture medium；The sense powdery mildew melon variety is the melon variety of high sense powdery mildew.
The water planting includes the following steps：
The seed for feeling powdery mildew melon variety is soaked seed 3.5 hours in 55 DEG C of warm water, is then placed in the training for being covered with wetting filter paper
It supports in ware, and covers culture dish lid；
Culture dish is placed under the dark surrounds of 29 DEG C of temperature to cultivate 20 hours and carries out vernalization；
After germination, culture dish is placed on 25 DEG C of temperature, 6000 lux of illuminance, 12 hours/12 hours photoperiod（Light
According to/dark）It is cultivated under environment；
When seedling height is close to culture dish height, opens culture dish lid and cultivated；
Growth of seedling situation is observed, supplements 1/2Hogland nutrient solutions into culture dish in time, keeps filter paper wetting until seedling
Rough leaf is fully deployed.
Step 4：Melon powdery mildew disease plant is chosen in muskmelon producing region, first with ear washing bulb by melon powdery mildew disease plant
The old spore on the powdery mildew scab surface on blade is blown off, is taken with oese within second day fresh in diameter 3mm powdery mildew scabs
Spore is inoculated on cotyledon described in step 3.
Step 5：Culture dish lid is covered after inoculation, and culture dish is placed on 20 DEG C of temperature, 2000 lux of illuminance, light
12 hours/12 hours period（Illumination/dark）It is cultivated under environment.
Step 6：After 37 days, transfer to the epicotyledonary powdery mildew germ.
The embodiment of the above, only presently preferred embodiments of the present invention, is only used to explain the present invention, not limit
The scope of the present invention processed to those of ordinary skill in the art certainly can be according to skill disclosed in this specification
Art content, makes other embodiments easily by way of replacing or changing, therefore all in the principle of the present invention and technique item
The changes and improvements etc. that part is done, should all be included in scope of the present invention patent.
Claims (4)
1. a kind of in-vitro conservation method of melon powdery mildew germ, which is characterized in that include the following steps：
Step 1：Prepare culture medium：In every 1000 milliliters of sterile waters be added 8 grams of agar, 6.8 grams of sucrose, 18.2 grams of mannitol and
The Natamycin solution of 400 microlitres of mass fractions 25%；Then culture medium is added in the culture dish after high pressure steam sterilization；
Step 2：After culture medium solidification, the blotting paper after 1~2 layer of high pressure steam sterilization is put on culture medium；
Step 3：The seed for feeling powdery mildew melon variety is subjected to water planting, after seedling grows the 1st true leaf, in superclean bench
On take the cotyledon of seedling, then will be sub with aseptic water washing 3 times after cotyledon is impregnated 3 seconds in the alcohol of volume fraction 75%
Leaf is lain in culture dish, and the lower surface of the cotyledon is downward；
Step 4：Melon powdery mildew disease plant is chosen in muskmelon producing region, first with ear washing bulb by melon powdery mildew disease plant blade
On the old spore on powdery mildew scab surface blow off, the Fresh spores in diameter 3mm powdery mildew scabs are taken with oese within second day,
It is inoculated on cotyledon described in step 3；
Step 5：Culture dish lid is covered after inoculation, and culture dish is placed on 20 DEG C of temperature, 2000 lux of illuminance, photoperiod
It is cultivated under 12 hours/12 hours environment；
Step 6：After 35~42 days, transfer to the epicotyledonary powdery mildew germ.
2. a kind of in-vitro conservation method of melon powdery mildew germ according to claim 1, which is characterized in that the water suction
There is circular hole, the petiole lower end of a diameter of 4~6mm of the circular hole, the cotyledon are located in circular hole, and are connect with culture medium on paper
It touches.
3. a kind of in-vitro conservation method of melon powdery mildew germ according to claim 1, which is characterized in that the culture
Culture medium thickness in ware is 2~4mm.
4. a kind of in-vitro conservation method of melon powdery mildew germ according to claim 1, which is characterized in that the sense is white
Powder disease melon variety is the melon variety of middle sense powdery mildew or high sense powdery mildew.
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Citations (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 |
| CN104293670A (en) * | 2014-10-27 | 2015-01-21 | 广西壮族自治区农业科学院蔬菜研究所 | Preservation method for momordica charantia powdery mildew |
| CN105255743A (en) * | 2015-10-26 | 2016-01-20 | 新疆农业科学院植物保护研究所 | Powdery mildew preserving and culturing device and method for preserving powdery mildew through device |
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2018
- 2018-03-20 CN CN201810231262.7A patent/CN108315260B/en active Active
Patent Citations (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 |
| CN104293670A (en) * | 2014-10-27 | 2015-01-21 | 广西壮族自治区农业科学院蔬菜研究所 | Preservation method for momordica charantia powdery mildew |
| CN105255743A (en) * | 2015-10-26 | 2016-01-20 | 新疆农业科学院植物保护研究所 | Powdery mildew preserving and culturing device and method for preserving powdery mildew through device |
Non-Patent Citations (1)
| Title |
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| 杜兴兰: "葡萄霜霉病和白粉病生物防治的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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