CN104293670A - Preservation method for momordica charantia powdery mildew - Google Patents
Preservation method for momordica charantia powdery mildew Download PDFInfo
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- CN104293670A CN104293670A CN201410583749.3A CN201410583749A CN104293670A CN 104293670 A CN104293670 A CN 104293670A CN 201410583749 A CN201410583749 A CN 201410583749A CN 104293670 A CN104293670 A CN 104293670A
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Abstract
The invention discloses a preservation method for momordica charantia powdery mildew. The preservation method comprises the following steps: inoculating powdery mildew on a diseased leaf into an in-vitro amenable-to-diseases momordica charantia leaf; picking out a single spore or a single spore stalk when a new powdery layer is generated on the leaf to be inoculated into an in-vitro leaf of an aseptic seedling for purification, reinoculating the purified single spore or the single spore stalk into a sterile momordica charantia cotyledon after a new colony is generated, and performing sealing for preservation after a new colony is generated on the surface of the cotyledon. Under the in vitro condition, by using the preservation method, momordica charantia powdery mildew can be preserved for more than 150 d; the purification and the preservation of the momordica charantia powdery mildew are realized under an artificial condition; the preservation method has the advantages of being small in occupied area, and controllable in preservation time, is used for identifying and controlling the momordica charantia powdery mildew, brings great convenience to selection of new varieties resistant to momordica charantia powdery mildew and biological research on the momordica charantia powdery mildew, and has a higher application value.
Description
Technical field
The invention belongs to the store method of a kind of obligate parasite in plant pathology technical field, especially relate to a kind of store method of balsam pear Powdery Mildew.
Background technology
Balsam pear (Momordica charantia L.), has another name called balsam pear, is the annual climbing herb plant of Curcurbitaceae.Balsam pear is a kind of vegetables that people like, country of origin China.In South East Asia, China and archipelago, the Caribbean Sea all have and plant widely.Along with the expansion gradually of balsam pear cultivated area and scale, balsam pear disease sickness rate is also day by day serious.
Balsam pear Powdery Mildew is one of common Major Diseases during balsam pear is produced, belong to climacteric disease, main in spring and autumn eruption and prevalence, because Powdery Mildew is a kind of obligate parasite, can only parasitize on live body host plant, can not cultivate on artificial medium, therefore be difficult under in vitro condition, carry out purifying and long-term preservation.Some purifying about balsam pear Powdery Mildew in prior art report and store method are mostly in Powdery Mildew Epidemic season in spring and autumn, Powdery Mildew is inoculated into and susceptible host plant carries out repeatedly singly preserving, general about one month of storage life single switching purifying.These class methods have by environment and seasonal effect, take up room large, the shortcoming that workload is large and the shelf time is short, simultaneously cause purity lower because the Powdery Mildew in field contains a large amount of miscellaneous bacteria, can not meet the demand of follow-up study work, such as Powdery Mildew qualification, molecular biology research, sterilant development etc. all need the live body pathogenic bacterium of a large amount of purifying.Therefore, in the urgent need to a kind of purifying and store method of efficient balsam pear Powdery Mildew.
Summary of the invention
The present invention can only parasitize the feature on live plant according to the Powdery Mildew of balsam pear, provide a kind of store method of balsam pear Powdery Mildew in vitro.
To achieve these goals, the present invention is achieved through the following technical solutions:
A kind of store method of balsam pear Powdery Mildew, that the powdery mildew of falling ill on leaf is inoculated into the in vitro momordica charantic leaf surface of easily susceptible health by field, be placed in 28 DEG C/16h, under 20 DEG C/8h alternation of light and darkness condition, moisturizing is cultivated, after producing new white powder layer, aseptically, choose single spore or wall scroll sporophore, purifying on the excised leaf being inoculated into aseptic seedling, be inoculated on aseptic balsam pear cotyledon after producing new bacterium colony, when cotyledon surface produces new bacterium colony, be placed in temperature 10 DEG C ~ 12 DEG C again, the condition lower seal of 8h/16h alternation of light and darkness is preserved, at in vitro, present method can preserve more than Powdery Mildew 150d.
Technical scheme provided by the present invention, its step is as follows:
(1) induction of balsam pear Powdery Mildew: first the powdery mildew on sick for balsam pear leaf is inoculated on healthy in vitro momordica charantic leaf, then in vitro momordica charantic leaf is put into culture dish, under sealing is placed in alternation of light and darkness condition, moisturizing is cultivated, and induces new Powdery Mildew;
(2) preparation of the aseptic blade of balsam pear and aseptic cotyledon: will be inoculated in substratum after the seed disinfection of easy susceptible material, obtains after aseptic seedling, the blade growing to wide about 3cm is cut to put into culture dish stand-by; Cut the stem section of band cotyledon from hypocotyl 1cm and remove lateral bud, insert every hole and fill in 12 porocytes cultivation plate holes of 3ml nutritive medium, the box with cover Tissue Culture Plate being put into sterilization is stand-by;
(3) purifying of balsam pear Powdery Mildew: aseptically, by Powdery Mildew newly-generated for induction, under anatomical lens, choose single spore or single sporophore, be inoculated on ready aseptic seedling excised leaf, sealing is placed in moisturizing culture purified under alternation of light and darkness condition;
(4) preservation of balsam pear Powdery Mildew: the Powdery Mildew after purifying is inoculated on the aseptic cotyledon that inserts and fill in the cell culture plate well of nutritive medium, again Tissue Culture Plate is put into the box with cover of sterilization, cultivate under sealing is placed in alternation of light and darkness condition, produce after new bacterium colony until cotyledon surface, then preserve under being placed in temperature 10 DEG C ~ 12 DEG C, the dark 8h/16h condition of light.
As preferably, blade described in step (1) is newborn on easy susceptible material side shoot 4th ~ 5 and be less than the young leaflet tablet of 9cm.
As preferably, described in step (1), (3), alternation of light and darkness condition is 28 DEG C/16h, 20 DEG C/8h, and intensity of illumination is 2000Lux.
As preferably, step (4) described alternation of light and darkness condition is temperature 10 DEG C ~ 12 DEG C, the dark 8h/16h of light, and intensity of illumination is 2000Lux.
As preferably, substratum described in step (2) is the MS substratum of additional sucrose 20g/L, agar 7g/L; Described nutritive medium is the 1/2MS substratum of additional benzoglyoxaline 100g/L, sucrose 15g/L, and wherein 1/2MS is the MS substratum that macroelement reduces by half.
beneficial effect of the present invention
1, the present invention utilizes the feature that balsam pear cotyledon life cycle is long, Powdery Mildew after purifying is preserved on balsam pear cotyledon, the restriction of having broken environment and having fallen ill to Powdery Mildew season, store method of the present invention has little, that the shelf time the is long advantage that takes up room.
2, the invention solves short problem of current balsam pear Powdery Mildew shelf time, be qualification and the control of balsam pear Powdery Mildew, seed selection and the biological study of balsam pear Powdery Mildew of balsam pear mildew-resistance new variety provide very big convenience, have higher using value.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
embodiment
1, for the vegetable material of examination
The balsam pear excised leaf of purifying Powdery Mildew is that field gathers newborn on easy susceptible material MC105 side shoot the 4th and be less than the healthy young leaflet tablet of 9cm; The aseptic excised leaf of purifying and the cotyledon preserved are obtained by easily susceptible balsam pear material MC105 planting seed in MS substratum.
2, implementation step
The collection of 2.1 balsam pear Powdery Mildews be separated
Field gathers the sick leaf that susceptible material MC105 still keeps green, powdery mildew is inoculated on healthy in vitro momordica charantic leaf, excised leaf is put into the culture dish that filter paper is housed, sealing is placed in 28 DEG C/16h, and under the condition of 20 DEG C/8h, the new Powdery Mildew of induction is cultivated in alternation of light and darkness moisturizing.Treat that 4d can be observed leaf surface and produces new fuzzy white powder layer, the white powder layer of 7th ~ 10d thickeies, and namely can be used for purifying.
The preparation of the aseptic blade of 2.2 balsam pear and aseptic cotyledon
Be inoculated in the MS substratum of additional sucrose 20g/L, agar 7g/L by after the seed disinfection of easy susceptible material MC105,5d seed germination, obtain aseptic seedling, during about 10d, the blade growing to wide about 3cm is cut that to put into the culture dish that aseptic filter paper is housed stand-by; Cut the stem section of band cotyledon from hypocotyl 1cm and remove lateral bud, insert every hole and fill in 12 porocytes cultivation plate holes of 3ml nutritive medium, not lid lid, the box with cover Tissue Culture Plate being put into sterilization is stand-by.
The purifying of 2.3 balsam pear Powdery Mildews
Aseptically by Powdery Mildew newly-generated for induction, under anatomical lens, choose single spore or wall scroll sporophore, be inoculated on aseptic seedling excised leaf, sealing is placed in 28 DEG C/16h, alternation of light and darkness moisturizing culture purified under the condition of 20 DEG C/8h, about 7d, the bacterium colony of macroscopic new generation.
The preservation of 2.4 balsam pear Powdery Mildews
The Powdery Mildew of purifying is inoculated on aseptic cotyledon, the Tissue Culture Plate of not lid lid is put into the box with cover of sterilization, sealing is placed on 28 DEG C/16h, under the condition of 20 DEG C/8h, alternation of light and darkness is cultivated, about 10d, cotyledon surface produces new bacterium colony, preserves under being then placed in temperature 10 DEG C ~ 12 DEG C, the dark 8h/16h condition of light.
3, balsam pear Powdery Mildew preservation effect
The cotyledon connecing bacterium is preserved under temperature 10 DEG C ~ 12 DEG C, the dark 8h/16h condition of light, 150d cotyledon still can keep more dark green, cotyledon now or the live body host plant of Powdery Mildew are described, 28 DEG C/16h is placed in connecing mushroom leaf, alternation of light and darkness restoration ecosystem 3d under the condition of 20 DEG C/8h, epicotyledonary powdery mildew is inoculated on healthy susceptible balsam pear material blade, states the same in front of causative effect, illustrate that the Powdery Mildew saving 150d on cotyledon still has stronger virulence.
The aforementioned description to concrete exemplary of the present invention is to illustrate and the object of illustration.These descriptions not want the present invention to be defined as disclosed precise forms, and obviously, according to above-mentioned instruction, can much change and change.The object selected exemplary embodiment and describe is to explain certain principles of the present invention and practical application thereof, thus those skilled in the art can be realized and utilize various different exemplary of the present invention and various different selection and change.Scope of the present invention is intended to limited by claims and equivalents thereof.
Claims (4)
1. a store method for balsam pear Powdery Mildew, its concrete steps are as follows:
(1) induction of balsam pear Powdery Mildew: first the powdery mildew on sick for balsam pear leaf is inoculated on healthy in vitro momordica charantic leaf, then in vitro momordica charantic leaf is put into culture dish, under sealing is placed in alternation of light and darkness condition, moisturizing is cultivated, and induces new Powdery Mildew;
(2) preparation of the aseptic blade of balsam pear and aseptic cotyledon: will be inoculated in substratum after the seed disinfection of easy susceptible material, obtains after aseptic seedling, the blade growing to wide about 3cm is cut to put into culture dish stand-by; Cut the stem section of band cotyledon from hypocotyl 1cm and remove lateral bud, insert every hole and fill in 12 porocytes cultivation plate holes of 3ml nutritive medium, the box with cover Tissue Culture Plate being put into sterilization is stand-by;
(3) purifying of balsam pear Powdery Mildew: under aseptic condition, by Powdery Mildew newly-generated for induction, under anatomical lens, choose single spore or wall scroll sporophore, be inoculated on ready aseptic seedling excised leaf, sealing is placed in moisturizing culture purified under alternation of light and darkness condition;
(4) preservation of balsam pear Powdery Mildew: the Powdery Mildew after purifying is inoculated on the aseptic cotyledon that inserts and fill in the cell culture plate well of nutritive medium, again Tissue Culture Plate is put into the box with cover of sterilization, cultivate under sealing is placed in alternation of light and darkness condition, produce after new bacterium colony until cotyledon surface, be placed in temperature 10 ~ 12 DEG C again, preserve under the dark 8h/16h condition of light.
2. store method as claimed in claim 1, is characterized in that: blade described in step (1) is newborn on easy susceptible material side shoot 4th ~ 5 and be less than the young leaflet tablet of 9cm.
3. store method as claimed in claim 1, is characterized in that: described in step (1) and (3), alternation of light and darkness condition is 28 DEG C/16h, 20 DEG C/8h; Alternation of light and darkness condition described in step (4) is then temperature 10 ~ 12 DEG C, and the dark 8h/16h of light, intensity of illumination is 2000Lux.
4. store method as claimed in claim 1, is characterized in that: substratum described in step (2) is the MS substratum of additional sucrose 20g/L, agar 7g/L; Described nutritive medium is the 1/2MS substratum of additional benzoglyoxaline 100g/L, sucrose 15g/L, and wherein 1/2MS is the MS substratum that macroelement reduces by half.
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| CN201410583749.3A CN104293670A (en) | 2014-10-27 | 2014-10-27 | Preservation method for momordica charantia powdery mildew |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446824A (en) * | 2017-08-21 | 2017-12-08 | 湖北省农业科学院植保土肥研究所 | A kind of single spore separation method of wheat powdery mildew |
| CN108315260A (en) * | 2018-03-20 | 2018-07-24 | 中国农业科学院郑州果树研究所 | A kind of in-vitro conservation method of melon powdery mildew germ |
| CN108690808A (en) * | 2018-05-29 | 2018-10-23 | 广东省农业科学院蔬菜研究所 | A kind of preservation of cucurbits powdery mildew bacterium and propagation method |
| CN113439763A (en) * | 2021-07-28 | 2021-09-28 | 广西壮族自治区农业科学院 | Application of endophytic fungus strain X22 in preventing and treating powdery mildew |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446824A (en) * | 2017-08-21 | 2017-12-08 | 湖北省农业科学院植保土肥研究所 | A kind of single spore separation method of wheat powdery mildew |
| CN107446824B (en) * | 2017-08-21 | 2019-12-17 | 湖北省农业科学院植保土肥研究所 | Single spore isolation method of erysiphe graminis |
| CN108315260A (en) * | 2018-03-20 | 2018-07-24 | 中国农业科学院郑州果树研究所 | A kind of in-vitro conservation method of melon powdery mildew germ |
| CN108315260B (en) * | 2018-03-20 | 2019-12-13 | 中国农业科学院郑州果树研究所 | In-vitro preservation method for melon powdery mildew germs |
| CN108690808A (en) * | 2018-05-29 | 2018-10-23 | 广东省农业科学院蔬菜研究所 | A kind of preservation of cucurbits powdery mildew bacterium and propagation method |
| CN113439763A (en) * | 2021-07-28 | 2021-09-28 | 广西壮族自治区农业科学院 | Application of endophytic fungus strain X22 in preventing and treating powdery mildew |
| CN113439763B (en) * | 2021-07-28 | 2022-05-10 | 广西壮族自治区农业科学院 | Application of endophytic fungus strain X22 in preventing and treating powdery mildew |
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