CN108315260B - In-vitro preservation method for melon powdery mildew germs - Google Patents
In-vitro preservation method for melon powdery mildew germs Download PDFInfo
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Abstract
the invention belongs to the technical field of biological pathogenic bacteria preservation, and particularly relates to an in vitro preservation method of melon powdery mildew pathogen. The method comprises the following steps: step 1: preparing a culture medium, and then adding the culture medium into a culture dish subjected to high-pressure steam sterilization; step 2: after the culture medium is solidified, 1-2 layers of absorbent paper are placed on the culture medium. And step 3: carrying out water culture on seeds of powdery mildew-susceptible melon varieties, taking cotyledons of seedlings after the 1 st true leaves of the seedlings grow out, and then flatly placing the disinfected cotyledons on absorbent paper; and 4, step 4: inoculating the cotyledons with powdery mildew germ. And 5: after inoculation, a cover of the culture dish is covered, and the culture dish is placed in an environment with the temperature of 20 ℃, the illumination intensity of 2000 luxes and the photoperiod (day/night) of 12 hours/12 hours for culture; step 6: and after 35-42 days, transferring powdery mildew germs on the cotyledons. The method has simple operation, low cost, and long germ storage time.
Description
Technical Field
the invention belongs to the technical field of biological pathogenic bacteria preservation, and particularly relates to an in vitro preservation method of melon powdery mildew pathogen.
background
the melon is of the family CucurbitaceaeCucurbitaceae) Cucumber genus (A)Cucumisl.) annual vining herbaceous plants. The China has a long history of cultivation of the melons and is the biggest producing and consuming nation of the melons in the world. According to FAO statistics, the planting area of the muskmelon in 2014 reaches 43.89 ten thousand hm2yield ofAccounts for 50.04% of the total world production. However, melon diseases have become more serious with the recent expansion of melon cultivation areas in protected areas. Powdery mildew is one of important diseases in melon production in a protected area, mainly infests plant leaves in the seedling stage and the adult stage of the melon, and also infests stems and fruits in a severe stage. Under a proper environmental condition, powdery mildew develops very quickly, and can spread to the whole greenhouse or melon field within a few days, so that leaves are yellow and dry, photosynthesis of plants is seriously influenced, and the yield and quality of melon fruits are further influenced.
In view of the serious hazards of melon powdery mildew, it has become a hot spot for researchers to study. However, melon powdery mildew, which belongs to obligate parasitic bacteria, either as mycelium or conidia in vegetative stage, has a short duration of viability after leaving the living host, and therefore powdery mildew must be preserved in vivo. In the current research of melon powdery mildew, in order to guarantee the annual preservation of powdery mildew, the researchers usually need to continuously culture infected plant seedlings, inoculate the seedlings by spraying spore suspension, and continuously transfer the germs. Although it has also been reported that when the plant grows to 5 true leaves, the stem tip is cut and inserted into a triangular flask containing a culture medium of benzimidazole-mannitol agarose, and then inoculated and preserved on the stem tip. The stem tip can be taken only when the plant grows to 5-6 leaves, so that the time and labor are wasted, the cost is high, and the stem tip is easy to pollute in the culture process. According to the traditional spore suspension liquid spray inoculation preservation method, seedling must be raised, inoculation is carried out when 3-4 true leaves grow on a plant, the leaves become yellow and dry quickly after the plant is diseased, the seedling raising cost is high, and the preservation time is short.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the in vitro preservation method of the melon powdery mildew pathogen, and the method greatly shortens the culture time of early-stage melon seedlings by in vitro inoculation of cotyledons, and has simple and convenient operation and low cost; meanwhile, most of nutrition is stored in the cotyledon after the seed germinates, so that the seed is inoculated with the cotyledon to store the germ for a longer time compared with the true leaf; in addition, the isolation of the cotyledon and the culture medium by the absorbent paper reduces the pollution probability.
In order to achieve the purpose, the invention adopts the following technical scheme:
An in vitro preservation method of melon powdery mildew germs comprises the following steps:
Step 1: preparing a culture medium: adding 8 g of agar, 6.8 g of cane sugar, 18.2 g of mannitol and 400 microliters of natamycin solution with the mass fraction of 25 percent into each 1000 milliliters of sterile water; the medium was then added to the autoclaved petri dish.
Step 2: and after the culture medium is solidified, 1-2 layers of water absorption paper subjected to high-pressure steam sterilization are placed on the culture medium.
and step 3: carrying out water culture on seeds of powdery mildew melon varieties, taking cotyledons of seedlings on a superclean workbench after the seedlings grow to 1 st true leaves, soaking the cotyledons in 75% alcohol by volume percentage for 3 seconds, washing the cotyledons with sterile water for 3 times, and then flatly placing the cotyledons in a culture dish, wherein the lower surfaces of the cotyledons face downwards.
The water culture comprises the following steps:
soaking seeds of powdery mildew melon varieties in warm water at 55 ℃ for 3-4 hours, then putting the seeds into a culture dish paved with wetting filter paper, and covering a culture dish cover;
Placing the culture dish in a dark environment at the temperature of 28-30 ℃ for culturing for 18-24 hours to accelerate germination;
After the seeds germinate, putting the culture dish in an environment with the temperature of 25 ℃, the illumination of 6000 luxes and the photoperiod of 12 hours/12 hours (light/dark) for culture;
opening the cover of the culture dish for culturing when the height of the seedling is close to that of the culture dish;
And observing the growth condition of the seedlings, supplementing 1/2 Hogland nutrient solution into the culture dish in time, and keeping the filter paper wet until the first true leaves of the seedlings are completely unfolded.
And 4, step 4: and (3) selecting a melon powdery mildew disease-susceptible plant in a melon production area, blowing off old spores on the surface of powdery mildew disease spots on leaves of the melon powdery mildew disease-susceptible plant by using an ear washing ball, taking fresh spores with the diameter of 3mm in the powdery mildew disease spots by using an inoculating loop on the next day, and inoculating the fresh spores to the cotyledon in the step (3).
And 5: after inoculation, the cover of the dish was closed, and the dish was incubated at 20 ℃ under a light intensity of 2000 luxes with a photoperiod of 12 hours/12 hours (light/dark).
step 6: and after 35-42 days, transferring powdery mildew germs on the cotyledons.
Furthermore, circular holes are formed in the absorbent paper, the diameter of each circular hole is 4-6 mm, and the lower ends of the petioles of the cotyledons are located in the circular holes and are in contact with the culture medium.
Further, the thickness of the culture medium in the culture dish is 2-4 mm.
Further, the powdery mildew-sensitive melon variety is a powdery mildew-sensitive melon variety or a powdery mildew-sensitive melon variety with high sensitivity.
compared with the prior art, the invention has the beneficial effects that:
1. According to the method, powdery mildew is inoculated on the in vitro cotyledon of the melon to preserve the in vitro cotyledon, and the powdery mildew can be inoculated after two cotyledons are completely unfolded and the first true leaf grows out; and the cotyledon has more nutrition than the true leaf, so the preservation time is longer.
2. The natamycin is added into the culture medium used by the invention, so that the culture medium can be effectively prevented from being polluted by other bacteria and actinomycetes, and in addition, the water absorption paper is covered on the culture medium, so that the large-area contact between cotyledons and the culture medium is avoided, and the pollution probability is further reduced.
3. in the optimized scheme of the invention, the water absorption paper is provided with the round hole, the lower end of the petiole of the cotyledon is positioned in the round hole, so that the cotyledon can directly absorb nutrition, and in addition, the leaf part of the cotyledon is positioned on the water absorption paper and is not contacted with a culture medium, so that the pollution probability is low.
4. The method of the invention uses the culture dish to store the powdery mildew, the using amount of the culture medium is less, and the cost is saved.
5. The method is mainly used for short-term preservation of powdery mildew, meets the requirement of using the powdery mildew in a short time, and has the advantages of short culture period, very simple and convenient operation and low cost.
drawings
FIG. 1 is a graph showing the in vitro preservation of powdery mildew fungus of Cucumis melo in example 2 of the present invention.
Detailed Description
the following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
example 1
An in vitro preservation method of melon powdery mildew germs comprises the following steps:
step 1: preparing a culture medium: adding 8 g of agar, 6.8 g of sucrose, 18.2 g of mannitol and 400 microliters of a natamycin solution with a mass fraction of 25% to 1000 milliliters of sterile water; the medium was then added to the autoclaved petri dish.
step 2: after the culture medium solidified, 1 layer of water-absorbent paper after high-pressure steam sterilization was placed on the culture medium.
And step 3: carrying out water culture on seeds of powdery mildew melon varieties, taking cotyledons of seedlings on an ultra-clean workbench after the 1 st true leaves of the seedlings grow out, soaking the cotyledons in alcohol with the volume percentage of 75% for 3 seconds, washing the cotyledons for 3 times by using sterile water, and then flatly placing the cotyledons on water absorption paper in a culture dish, wherein the lower surfaces of the cotyledons face downwards; the powdery mildew-susceptible melon variety is a powdery mildew-susceptible melon variety.
The water culture comprises the following steps:
Soaking seeds of powdery mildew melon varieties in warm water at 55 ℃ for 3 hours, then putting the seeds into a culture dish paved with wetting filter paper, and covering a culture dish cover;
Culturing the culture dish in a dark environment at the temperature of 28 ℃ for 24 hours to accelerate germination;
After the seeds germinate, putting the culture dish in an environment with the temperature of 25 ℃, the illumination of 6000 luxes and the photoperiod of 12 hours/12 hours (light/dark) for culture;
opening the cover of the culture dish for culturing when the height of the seedling is close to that of the culture dish;
And observing the growth condition of the seedlings, supplementing 1/2 Hogland nutrient solution into the culture dish in time, and keeping the filter paper wet until the first true leaves of the seedlings are completely unfolded.
And 4, step 4: and (3) selecting a melon powdery mildew disease-susceptible plant in a melon production area, blowing off old spores on the surface of powdery mildew disease spots on leaves of the melon powdery mildew disease-susceptible plant by using an ear washing ball, taking fresh spores with the diameter of 3mm in the powdery mildew disease spots by using an inoculating loop on the next day, and inoculating the fresh spores to the cotyledon in the step (3).
and 5: after inoculation, the cover of the dish was closed, and the dish was incubated at 20 ℃ under a light intensity of 2000 luxes with a photoperiod of 12 hours/12 hours (light/dark).
Step 6: after 35 days, the powdery mildew germs on the cotyledons are transferred.
Example 2
An in vitro preservation method of melon powdery mildew germs comprises the following steps:
step 1: preparing a culture medium: adding 8 g of agar, 6.8 g of sucrose and 18.2 g of mannitol to 1000 ml of sterile water, then heating until the agar melts, and then adding 400 microliters of a natamycin solution with a mass fraction of 25% before the medium solidifies; the medium was then added to the autoclaved dish, the thickness of which was 4 mm.
step 2: after the culture medium is solidified, 1 layer of water absorption paper after high-pressure steam sterilization is placed on the culture medium, and circular holes are formed in the water absorption paper and are 6mm in diameter.
and step 3: carrying out water culture on seeds of powdery mildew melon varieties, taking cotyledons of seedlings on an ultra-clean workbench after the 1 st true leaves of the seedlings grow out, soaking the cotyledons in alcohol with the volume fraction of 75% for 3 seconds, washing the cotyledons for 3 times by using sterile water, then flatly placing the cotyledons on water absorption paper in a culture dish, enabling the lower surfaces of the cotyledons to face downwards, and enabling the lower ends of petioles of the cotyledons to be located in the round holes in the step 2 and to be in contact with a culture medium; the powdery mildew-susceptible melon variety is a powdery mildew-susceptible melon variety.
the water culture comprises the following steps:
Soaking seeds of powdery mildew melon varieties in warm water at 55 ℃ for 4 hours, then putting the seeds into a culture dish paved with wetting filter paper, and covering a culture dish cover;
culturing the culture dish in a dark environment at the temperature of 30 ℃ for 18 hours to accelerate germination;
After the seeds germinate, putting the culture dish in an environment with the temperature of 25 ℃, the illumination of 6000 luxes and the photoperiod of 12 hours/12 hours (light/dark) for culture;
Opening the cover of the culture dish for culturing when the height of the seedling is close to that of the culture dish;
and observing the growth condition of the seedlings, supplementing 1/2 Hogland nutrient solution into the culture dish in time, and keeping the filter paper wet until the first true leaves of the seedlings are completely unfolded.
And 4, step 4: and (3) selecting a melon powdery mildew disease-susceptible plant in a melon production area, blowing off old spores on the surface of powdery mildew disease spots on leaves of the melon powdery mildew disease-susceptible plant by using an ear washing ball, taking fresh spores with the diameter of 3mm in the powdery mildew disease spots by using an inoculating loop on the next day, and inoculating the fresh spores to the cotyledon in the step (3).
and 5: after inoculation, the cover of the dish was closed, and the dish was incubated at 20 ℃ under a light intensity of 2000 luxes with a photoperiod of 12 hours/12 hours (light/dark).
Step 6: after 42 days, the cotyledons were inoculated with powdery mildew.
Example 3
An in vitro preservation method of melon powdery mildew germs comprises the following steps:
step 1: preparing a culture medium: 16 g of agar, 13.6 g of sucrose and 36.4 g of mannitol were added to 2000 ml of sterile water, which was then heated until the agar melted, followed by addition of 800. mu.l of a 25% by mass natamycin solution before the medium solidified; the medium was then added to the autoclaved dish, the thickness of which was 3 mm.
Step 2: after the culture medium is solidified, 1 layer of water absorption paper after high-pressure steam sterilization is placed on the culture medium, and circular holes are formed in the water absorption paper and are 5mm in diameter.
And step 3: carrying out water culture on seeds of powdery mildew melon varieties, taking cotyledons of seedlings on an ultra-clean workbench after the 1 st true leaves of the seedlings grow out, soaking the cotyledons in alcohol with the volume fraction of 75% for 3 seconds, washing the cotyledons for 3 times by using sterile water, then flatly placing the cotyledons on water absorption paper in a culture dish, enabling the lower surfaces of the cotyledons to face downwards, and enabling the lower ends of petioles of the cotyledons to be located in the round holes in the step 2 and to be in contact with a culture medium; the powdery mildew-susceptible melon variety is a powdery mildew-susceptible melon variety; the hydroponic procedure was as in example 2.
and 4, step 4: and (3) selecting a melon powdery mildew disease-susceptible plant in a melon production area, blowing off old spores on the surface of powdery mildew disease spots on leaves of the melon powdery mildew disease-susceptible plant by using an ear washing ball, taking fresh spores with the diameter of 3mm in the powdery mildew disease spots by using an inoculating loop on the next day, and inoculating the fresh spores to the cotyledon in the step (3).
And 5: after inoculation, the cover of the dish was closed, and the dish was incubated at 20 ℃ under a light intensity of 2000 luxes with a photoperiod of 12 hours/12 hours (light/dark).
Step 6: after 40 days, the powdery mildew germs on the cotyledons are transferred.
example 4
An in vitro preservation method of melon powdery mildew germs comprises the following steps:
Step 1: preparing a culture medium: adding 8 g of agar, 6.8 g of sucrose and 18.2 g of mannitol to 1000 ml of sterile water, then heating until the agar melts, and then adding 400 microliters of a natamycin solution with a mass fraction of 25% before the medium solidifies; the medium was then added to the autoclaved dish, the thickness of which was 2 mm.
Step 2: after the culture medium is solidified, 2 layers of water absorption paper after high-pressure steam sterilization are placed on the culture medium, and circular holes are formed in the water absorption paper and are 4mm in diameter.
And step 3: carrying out water culture on seeds of powdery mildew melon varieties, taking cotyledons of seedlings on an ultra-clean workbench after the 1 st true leaves of the seedlings grow out, soaking the cotyledons in alcohol with the volume fraction of 75% for 3 seconds, washing the cotyledons for 3 times by using sterile water, then flatly placing the cotyledons on water absorption paper in a culture dish, enabling the lower surfaces of the cotyledons to face downwards, and enabling the lower ends of petioles of the cotyledons to be located in the round holes in the step 2 and to be in contact with a culture medium; the powdery mildew-susceptible melon variety is a powdery mildew-susceptible melon variety.
The water culture comprises the following steps:
Soaking seeds of powdery mildew melon varieties in warm water at 55 ℃ for 3.5 hours, then putting the seeds into a culture dish paved with wetting filter paper, and covering a culture dish cover;
Culturing the culture dish in a dark environment at the temperature of 29 ℃ for 20 hours to accelerate germination;
After the seeds germinate, putting the culture dish in an environment with the temperature of 25 ℃, the illumination of 6000 luxes and the photoperiod of 12 hours/12 hours (light/dark) for culture;
Opening the cover of the culture dish for culturing when the height of the seedling is close to that of the culture dish;
And observing the growth condition of the seedlings, supplementing 1/2 Hogland nutrient solution into the culture dish in time, and keeping the filter paper wet until the first true leaves of the seedlings are completely unfolded.
And 4, step 4: and (3) selecting a melon powdery mildew disease-susceptible plant in a melon production area, blowing off old spores on the surface of powdery mildew disease spots on leaves of the melon powdery mildew disease-susceptible plant by using an ear washing ball, taking fresh spores with the diameter of 3mm in the powdery mildew disease spots by using an inoculating loop on the next day, and inoculating the fresh spores to the cotyledon in the step (3).
And 5: after inoculation, the cover of the dish was closed, and the dish was incubated at 20 ℃ under a light intensity of 2000 luxes with a photoperiod of 12 hours/12 hours (light/dark).
Step 6: after 37 days, the powdery mildew germs on the cotyledons are transferred.
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily implemented by those skilled in the art by means of replacement or modification according to the technical contents disclosed in the specification, and therefore, all changes and modifications that come within the spirit and technical conditions of the present invention should be included in the claims of the present invention.
Claims (3)
1. An in vitro preservation method of melon powdery mildew germs is characterized by comprising the following steps:
Step 1: preparing a culture medium: adding 8 g of agar, 6.8 g of cane sugar, 18.2 g of mannitol and 400 microliters of natamycin solution with the mass fraction of 25 percent into each 1000 milliliters of sterile water; then adding the culture medium into a culture dish sterilized by high-pressure steam;
step 2: after the culture medium is solidified, 1-2 layers of water absorption paper after high-pressure steam sterilization are placed on the culture medium;
The water absorption paper is provided with round holes, and the diameter of each round hole is 4-6 mm;
and step 3: carrying out water culture on seeds of powdery mildew melon varieties, taking cotyledons of seedlings on an ultra-clean workbench after the 1 st true leaves of the seedlings grow out, soaking the cotyledons in alcohol with the volume fraction of 75% for 3 seconds, washing the cotyledons for 3 times by using sterile water, and then flatly placing the cotyledons in a culture dish with the lower surfaces of the cotyledons facing downwards; the lower end of the petiole of the cotyledon is positioned in the round hole and is contacted with the culture medium;
And 4, step 4: selecting a melon powdery mildew disease-susceptible plant in a melon production area, blowing off old spores on the surface of powdery mildew disease spots on leaves of the melon powdery mildew disease-susceptible plant by using an ear washing ball, taking fresh spores with the diameter of 3mm in a inoculating loop on the next day, and inoculating the fresh spores onto the cotyledon in the step 3;
and 5: after inoculation, a cover of the culture dish is covered, and the culture dish is placed in an environment with the temperature of 20 ℃, the illumination intensity of 2000 luxes and the photoperiod of 12 hours/12 hours for culture;
step 6: and after 35-42 days, transferring powdery mildew germs on the cotyledons.
2. the in vitro preservation method of powdery mildew fungus of melon, according to claim 1, characterized in that the thickness of the culture medium in said culture dish is 2-4 mm.
3. The method for in vitro preservation of powdery mildew fungus of melon according to claim 1, wherein said powdery mildew-sensitive melon variety is a powdery mildew-sensitive or powdery mildew-susceptible melon variety.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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| 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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