CN104726349A - Tobacco phytophthora culture medium and preparation method thereof - Google Patents
Tobacco phytophthora culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a tobacco phytophthora culture medium and a preparation method thereof. The tobacco phytophthora culture medium is prepared by phytophgous substance, CaCO3 and agar, and comprises, calculated by 100 mL, 80-100 mL of liquid prepared by the phytophgous substance, 0.2-0.4 g of CaCO3 and 1.7-2.5 g of agar, water is used as mixture agent of the culture medium and is mixed to rated quantity, the pH value of the tobacco phytophthora culture medium is 6.8 to 7.0. The preparation method includes steps of pre-treatment, preparation of culture medium and after-treatment, to be more specifically, slicing 20-40 g of pumpkin (towel gourd, lotus root or sweet potato), adding it into 100 mL of water, smashing and filtering to remove residues, replenishing water to 100 mL, adding 0.2-0.4 g of CaCO3 and 1.7-2.5 g of agar and sterilizing. The culture medium is good in tobacco phytophthora effect, hypha is fast in growth and spore production is high. Culture time can be saved so as to save culture time and space, reduce pollution and energy consumption of culture, and culture efficiency is improved. The tobacco phytophthora culture medium is wide in raw material sources and low in cost, and the preparation method is simple, easy to learn and worthy of popularization and application.
Description
Technical field
The invention belongs to field of microbial culture technology, be specifically related to a kind of Phytophthora nicotianae substratum and preparation method thereof.
Background technology
Black shank be by Phytophthora nicotianae (
phytophthora nicotianae) the one soil that causes passes oomycetes disease, is the important disease affecting tobacco production and quality.Under natural condition, this disease infects primarily of the zoospore of its pathogenic bacteria Phytophthora nicotianae, propagates.For controlling this disease, need to carry out systematic research to pathogenic bacteria Phytophthora nicotianae, it is an important job that the wherein cultivation of Phytophthora nicotianae, the generation of zoospore and release etc. are studied, and is also tobacco black shank resistance qualification, pesticide control screening, Pathogen Biologic Characteristic and most basic in the aspect research such as mutual work between pathogenic bacteria and host.Research both domestic and external shows, the cultivation of Phytophthora nicotianae, the generation of zoospore and release are closely related with culture condition, and in these culture condition, the matrix of cultivation and substratum are crucial conditions.Much more external adopt V8 substratum, domestic adopt oat medium more, the growth of these 2 kinds of substratum, sporulation capacity often can not meet the needs that zoospore uses.Therefore, the substratum researching and developing culture efficiency higher becomes the task of top priority.
Summary of the invention
The first object of the present invention is to provide a kind of Phytophthora nicotianae substratum; Second object is to provide the preparation method of described Phytophthora nicotianae substratum.
The first object of the present invention is achieved in that with phytophagy material, CaCO
3be that raw material prepares with agar, in 100mL, containing the juice 80 ~ 100mL prepared with phytophagy material, CaCO
30.2 ~ 0.4g and agar 1.7 ~ 2.5g, using water as substratum blender, allotment is to specified rate, and described Phytophthora nicotianae Medium's PH Value is 6.8 ~ 7.0.
The second object of the present invention is achieved in that and comprises pre-treatment, medium preparing, post-processing step, specifically comprises:
A, pre-treatment: get phytophagy material peeling section, add the water of solid-liquid volume ratio 2 ~ 5 times, smash to pieces, filter and remove residue, filtrate moisturizing to specified rate obtains phytophagy material juice;
B, medium preparing: get phytophagy material juice, CaCO by formulation ratio respectively
3, adjust ph is 6.8 ~ 7.0, adds the agar of formulation ratio, obtains target substratum after heating makes to dissolve completely;
C, aftertreatment: target substratum is carried out packing, obtain finished product Phytophthora nicotianae substratum through sterilising treatment.
Culture medium culturing Phytophthora nicotianae of the present invention is effective, and mycelial growth rate is fast, sporulation capacity is large; Incubation time is short can save the energy consumption of the Time and place of cultivation, decreasing pollution and cultivation, improves the efficiency of cultivation; Making raw material sources of the present invention are extensive, with low cost, and making method is easy to learn, is worthy of popularization.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but limited the present invention never in any form, and any conversion done based on training centre of the present invention or replacement, all belong to protection scope of the present invention.
Phytophthora nicotianae substratum of the present invention is with phytophagy material, CaCO
3be that raw material prepares with agar, in 100mL, containing the juice 80 ~ 100mL prepared with phytophagy material, CaCO
30.2 ~ 0.4g and agar 1.7 ~ 2.5g, using water as substratum blender, allotment is to specified rate, and described Phytophthora nicotianae Medium's PH Value is 6.8 ~ 7.0.
Described phytophagy material is pumpkin, sponge gourd, lotus root or Ipomoea batatas, is preferably pumpkin or Ipomoea batatas.
20 ~ 40g phytophagy material peeling section got by the juice that described phytophagy material prepares, and add 40 ~ 80mL water, smash to pieces, filter and remove residue, filtrate moisturizing obtains to 100mL.
The preparation method of Phytophthora nicotianae substratum of the present invention, comprises pre-treatment, medium preparing, post-processing step, specifically comprises:
A, pre-treatment: get phytophagy material peeling section, add the water of solid-liquid volume ratio 2 ~ 5 times, smash to pieces, filter and remove residue, filtrate moisturizing to specified rate obtains phytophagy material juice;
B, medium preparing: get phytophagy material juice, CaCO by formulation ratio respectively
3, adjust ph is 6.8 ~ 7.0, adds the agar of formulation ratio, obtains target substratum after heating makes to dissolve completely;
C, aftertreatment: target substratum is carried out packing, obtain finished product Phytophthora nicotianae substratum through sterilising treatment.
Smashing to pieces described in step A adopts tissue mashing machine to smash 30 ~ 50s to pieces.
Filter and remove residue described in step A takes to be no less than hospital gauze or the special filtering filtration of material of 2 layers.
Sterilising treatment described in step C takes the one in moist heat sterilization, high-temperature sterilization or steam instantaneous sterilizing.
Sterilising treatment described in step C is high-temperature sterilization 20 ~ 40min at taking 121 DEG C.
The preparation method of Phytophthora nicotianae substratum of the present invention, concrete operations are as follows:
A, by 20 ~ 40g phytophagy material peeling section, add in 100mL water, smash to pieces, filter and remove residue, supply water and be settled to 100mL, mixing, namely obtain 100mL phytophagy material and smash liquid to pieces;
B, to smash to pieces in liquid at the phytophagy material of steps A and add CaCO
30.2 ~ 0.4g, agar 1.7 ~ 2.5g, 121 DEG C of sterilizings 15 ~ 30 minutes, are cooled to 50 ~ 60 DEG C, shake up, be down flat plate.
Embodiment 1
1, materials and methods
1.1 material
1.1.1 for examination bacterial classification
Phytophthora nicotianae No. 0 physiological strain Pn 119 bacterial strain.
1.1.2 substratum
Substratum: oat medium, 10% V
8substratum, pumpkin substratum, sponge gourd substratum, lotus root substratum, Ipomoea batatas substratum.
1.2 method
1.2.1 the preparation of substratum
Pumpkin substratum: 20 ~ 40g pumpkin is cleaned peeling, cut into pieces, and add deionized water 50mL, smash about 40s to pieces with tissue mashing machine, 2 layers of filtered through gauze remove slag, and supply water to 100mL, add agar 2g, CaCO
30.4g.The preparation of all the other substratum is with pumpkin substratum.Each substratum, before adding agar, regulates its pH, and the pH of substratum is remained between 6.8 ~ 7.0.
The substratum packing 250mL triangular flask 121 DEG C prepared, 20min sterilizing, treat that substratum temperature is down to about 50 DEG C, and substratum is poured into by Bechtop in diameter 9cm sterilizing culture dish, each culture dish 20mL substratum, cools for subsequent use.
1.2.2 growth medium is selected
Phytophthora nicotianae activates 2 ~ 3 times on oat medium flat board, and 28 DEG C of dark culturing 7 ~ 10 days, beat the mycelia block of cut-off footpath 5mm by donut with punch tool, and tweezers pipette a ferfas silk face and amplexiform to each on examination culture medium flat plate.That is often kind of substratum 5 flat boards repeat for 5 times, 28 DEG C of dark culturing.Cultivate and measure day growth speed on the 3rd, 5 day, record hyphae length (mycelia thin and thick density), cover with dull and stereotyped number of days.
1.2.3 produce spore ability to measure
After cultivating 10 days for examination culture medium inoculated, punch tool beats cut-off footpath 5mm mycelia block 15 pieces in the empty culture dish of sterilizing, adopts mycelia clump water culture induction inducing spore capsule, alternating temperature release zoospore, counting zoospore number.
2, results and analysis
2.1 the selection of growth medium
In 6 kinds of substratum for examination, all substratum all can grow, colony colour is pure white, but there is significant difference in mycelial growth rate, what the speed of growth was the fastest is Ipomoea batatas substratum, pumpkin substratum, next is oat medium, and be sponge gourd substratum, lotus root substratum again, that worst is 10% V
8substratum (table 1).From covering with the dull and stereotyped time, 10% V
8more late than other substratum 3 days of substratum, and other just can cover with flat board in 6 days.
The growing state of table 1 Phytophthora nicotianae on different culture media
2.2 produce spore ability measures
In 6 kinds of substratum, produce spore capacity variance significantly, sponge gourd substratum > Ipomoea batatas substratum > lotus root substratum > pumpkin substratum > oat medium >10% V
8substratum, 10% V
8there is the difference (table 2) on the order of magnitude in substratum and other substratum.
Table 2 Phytophthora nicotianae produces spore ability and compares (induction of 10%V8 nutrient solution)
2.3 Integrated comparative
In 6 kinds of substratum, Integrated comparative colony growth rate and product spore ability, can find Ipomoea batatas substratum, pumpkin substratum is the speed of growth and produces spore ability to be all better than general oat medium, be the substitutive medium that efficiency is higher; Although sponge gourd substratum, the lotus root substratum speed of growth are lower than oat medium, produce spore ability and be significantly higher than oat medium, overall efficiency is better than oat medium.10%V
8the culture medium culturing time is longer, and overall efficiency is low, is not suitable for cultivating Phytophthora nicotianae mycelia.
Claims (8)
1. a Phytophthora nicotianae substratum, is characterized in that described Phytophthora nicotianae substratum is with phytophagy material, CaCO
3be that raw material prepares with agar, in 100mL, containing the juice 80 ~ 100mL prepared with phytophagy material, CaCO
30.2 ~ 0.4g and agar 1.7 ~ 2.5g, using water as substratum blender, allotment is to specified rate, and described Phytophthora nicotianae Medium's PH Value is 6.8 ~ 7.0.
2. Phytophthora nicotianae substratum according to claim 1, is characterized in that described phytophagy material is pumpkin, sponge gourd, lotus root or Ipomoea batatas, is preferably pumpkin or Ipomoea batatas.
3. Phytophthora nicotianae substratum according to claim 1, it is characterized in that 20 ~ 40g phytophagy material peeling section got by the juice that described phytophagy material prepares, add 40 ~ 80mL water, smash to pieces, filter and remove residue, filtrate moisturizing obtains to 100mL.
4. a preparation method for the arbitrary described Phytophthora nicotianae substratum of claim 1 ~ 3, is characterized in that comprising pre-treatment, medium preparing, post-processing step, specifically comprises:
A, pre-treatment: get phytophagy material peeling section, add the water of solid-liquid volume ratio 2 ~ 5 times, smash to pieces, filter and remove residue, filtrate moisturizing to specified rate obtains phytophagy material juice;
B, medium preparing: get phytophagy material juice, CaCO by formulation ratio respectively
3, adjust ph is 6.8 ~ 7.0, adds the agar of formulation ratio, obtains target substratum after heating makes to dissolve completely;
C, aftertreatment: target substratum is carried out packing, obtain finished product Phytophthora nicotianae substratum through sterilising treatment.
5. preparation method according to claim 4, is characterized in that smashing to pieces described in step A adopts tissue mashing machine to smash 30 ~ 50s to pieces.
6. preparation method according to claim 4, is characterized in that the filter and remove residue described in step A takes to be no less than hospital gauze or the special filtering filtration of material of 2 layers.
7. preparation method according to claim 4, is characterized in that the sterilising treatment described in step C takes the one in moist heat sterilization, high-temperature sterilization or steam instantaneous sterilizing.
8. the preparation method according to claim 4 or 7, is characterized in that the sterilising treatment described in step C is high-temperature sterilization 20 ~ 40min at taking 121 DEG C.
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Cited By (3)
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| CN104946545A (en) * | 2015-07-14 | 2015-09-30 | 云南省烟草农业科学研究院 | Phytophthora nicotianae culturing method |
| CN108410793A (en) * | 2018-04-27 | 2018-08-17 | 江西省农业科学院植物保护研究所 | A kind of induction phytophthora blight of pepper production culture medium of spore, preparation method and application |
| CN108795778A (en) * | 2018-06-27 | 2018-11-13 | 江西省农业科学院植物保护研究所 | A kind of preparation method and application of Phytophthora capsici bacterium culture medium |
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| CN103045529A (en) * | 2012-12-31 | 2013-04-17 | 广东省农业科学院植物保护研究所 | Method of inducing phytophthora nicotianae breda de haan bacteria to generate sporangiums and release zoospores |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946545A (en) * | 2015-07-14 | 2015-09-30 | 云南省烟草农业科学研究院 | Phytophthora nicotianae culturing method |
| CN108410793A (en) * | 2018-04-27 | 2018-08-17 | 江西省农业科学院植物保护研究所 | A kind of induction phytophthora blight of pepper production culture medium of spore, preparation method and application |
| CN108410793B (en) * | 2018-04-27 | 2020-11-06 | 江西省农业科学院植物保护研究所 | Culture medium for inducing phytophthora capsici to produce spores, preparation method and application |
| CN108795778A (en) * | 2018-06-27 | 2018-11-13 | 江西省农业科学院植物保护研究所 | A kind of preparation method and application of Phytophthora capsici bacterium culture medium |
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