CN104726349B - A kind of Phytophthora nicotianae culture medium and preparation method thereof - Google Patents
A kind of Phytophthora nicotianae culture medium and preparation method thereof Download PDFInfo
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- CN104726349B CN104726349B CN201510174981.6A CN201510174981A CN104726349B CN 104726349 B CN104726349 B CN 104726349B CN 201510174981 A CN201510174981 A CN 201510174981A CN 104726349 B CN104726349 B CN 104726349B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention discloses a kind of Phytophthora nicotianae culture medium and preparation method thereof, the Phytophthora nicotianae culture medium is with phytophagous substance, CaCO3It is prepared with agar for raw material, in terms of 100mL, containing the juice 80 ~ 100mL, CaCO being prepared with phytophagous substance3 1.7 ~ 2.5g of 0.2 ~ 0.4g and agar is allocated using water as culture medium blender to rated capacity, and Phytophthora nicotianae Medium's PH Value is 6.8 ~ 7.0.The preparation method, including pre-treatment, culture medium preparation, post-processing step, specifically by 20 ~ 40g pumpkins（Sponge gourd, lotus root or sweet potato）Section is added in 100mL water, smashed to pieces, filter and remove residue, is supplied water to 100mL, is added in CaCO30.2 ~ 0.4g, agar 1.7 ~ 2.5g sterilizing, thus obtaining the products.Medium culture Phytophthora nicotianae effect of the present invention is good, and mycelial growth rate is fast, sporulation capacity is big；The incubation time short time that can save culture and space, the energy consumption for reducing pollution and culture improve the efficiency of culture；The making raw material sources of the present invention are extensive, of low cost, and production method is easy to learn, is worthy of popularization.
Description
Technical field
The invention belongs to field of microbial culture technology, and in particular to a kind of Phytophthora nicotianae culture medium and preparation method thereof.
Background technology
Tobacco black shank is by Phytophthora nicotianae（Phytophthora nicotianae) caused by it is a kind of soil pass oomycetes disease
Evil is the important disease for influencing tobacco production and quality.Under natural conditions, the disease is mainly by the travelling of its pathogen Phytophthora nicotianae
Spore is infected, propagated.The disease to pathogen Phytophthora nicotianae, it is necessary to carry out systematic research, wherein Phytophthora nicotianae in order to control
Culture, the generation of zoospore and the researchs such as release are an important job and tobacco black shank resistance identification, prevention medicine
It is most basic in interaction etc. research between agent screening, Pathogen Biologic Characteristic and pathogen and host.It is both domestic and external to grind
It is closely related with condition of culture to study carefully the culture for showing Phytophthora nicotianae, the generation of zoospore and release, in these condition of culture,
Matrix, that is, culture medium of culture is crucial condition.It is external mostly using V8 culture mediums, it is domestic mostly using oat medium, this 2 kinds
The growth of culture medium, sporulation capacity tend not to meet the needs that zoospore uses.Therefore, the higher training of culture efficiency is researched and developed
Supporting base becomes the task of top priority.
The content of the invention
The first object of the present invention is to provide a kind of Phytophthora nicotianae culture medium；Second is designed to provide the tobacco
The preparation method of Phytophthora culture medium.
The first object of the present invention is achieved in that with phytophagous substance, CaCO3It is prepared into agar for raw material
It arrives, in terms of 100mL, containing the juice 80 ~ 100mL, CaCO being prepared with phytophagous substance30.2 ~ 0.4g and agar 1.7 ~
2.5g is allocated using water as culture medium blender to rated capacity, and the Phytophthora nicotianae Medium's PH Value is 6.8 ~ 7.0.
The second object of the present invention is achieved in that including pre-treatment, culture medium preparation, post-processing step, specific to wrap
It includes：
A, pre-treatment：The peeling section of phytophagous substance is taken, the water of 2 ~ 5 times of solid-liquid volume ratio is added in, smashs to pieces, filter and remove residue,
Filtrate moisturizing to rated capacity obtains phytophagous substance juice；
B, prepared by culture medium：Respectively phytophagous substance juice, CaCO are taken by formulation ratio3, it is 6.8 ~ 7.0 to adjust pH value,
The agar of formulation ratio is added in, heating makes to obtain target culture medium after being completely dissolved；
C, post-process：Target culture medium is dispensed, sterilized processing finished product Phytophthora nicotianae culture medium.
The medium culture Phytophthora nicotianae effect of the present invention is good, and mycelial growth rate is fast, sporulation capacity is big；Incubation time is short
The time of culture and space, the energy consumption for reducing pollution and culture can be saved, improves the efficiency of culture；The making of the present invention is former
Material derives from a wealth of sources, is of low cost, and production method is easy to learn, is worthy of popularization.
Specific embodiment
With reference to embodiment, the present invention is further illustrated, but the present invention is not any limitation as in any way,
Based on present invention teach that any conversion or replacement made, all belong to the scope of protection of the present invention.
Phytophthora nicotianae culture medium of the present invention is with phytophagous substance, CaCO3It is prepared with agar for raw material,
In terms of 100mL, containing the juice 80 ~ 100mL, CaCO being prepared with phytophagous substance31.7 ~ 2.5g of 0.2 ~ 0.4g and agar,
It is allocated using water as culture medium blender to rated capacity, the Phytophthora nicotianae Medium's PH Value is 6.8 ~ 7.0.
The phytophagous substance is pumpkin, sponge gourd, lotus root or sweet potato, is preferably pumpkin or sweet potato.
The juice that the phytophagous substance is prepared is to take the peeling section of 20 ~ 40g phytophagous substance, addition 40 ~
80mL water, is smashed to pieces, filter and remove residue, and filtrate moisturizing to 100mL obtains.
The preparation method of Phytophthora nicotianae culture medium of the present invention, including pre-treatment, prepared by culture medium, post processing walks
Suddenly, specifically include：
A, pre-treatment：The peeling section of phytophagous substance is taken, the water of 2 ~ 5 times of solid-liquid volume ratio is added in, smashs to pieces, filter and remove residue,
Filtrate moisturizing to rated capacity obtains phytophagous substance juice；
B, prepared by culture medium：Respectively phytophagous substance juice, CaCO are taken by formulation ratio3, it is 6.8 ~ 7.0 to adjust pH value,
The agar of formulation ratio is added in, heating makes to obtain target culture medium after being completely dissolved；
C, post-process：Target culture medium is dispensed, sterilized processing finished product Phytophthora nicotianae culture medium.
Smashing to pieces described in step A is to smash 30 ~ 50s to pieces using tissue mashing machine.
Filter and remove residue described in step A is to take no less than 2 layers of hospital gauze or special filtering filtration of material.
Sterilization treatment described in step C is the one kind taken in moist heat sterilization, high-temperature sterilization or steam instantaneous sterilizing.
Sterilization treatment described in step C is to take 20 ~ 40min of high-temperature sterilization at 121 DEG C.
The preparation method of Phytophthora nicotianae culture medium of the present invention, concrete operations are as follows：
A, 20 ~ 40g phytophagous substance is removed the peel and cut into slices, added in 100mL water, smash to pieces, filter and remove residue, supply water and be settled to
100mL, mixing smash liquid to pieces to get to 100mL phytophagous substances；
B, CaCO is added in the phytophagous substance of step A smashs liquid to pieces30.2 ~ 0.4g, 1.7 ~ 2.5g of agar, 121 DEG C go out
Bacterium 15 ~ 30 minutes, is cooled to 50 ~ 60 DEG C, shakes up, be down flat plate.
Embodiment 1
1st, materials and methods
1.1 material
1.1.1 for examination strain
No. 0 119 bacterial strain of biological strain Pn of Phytophthora nicotianae.
1.1.2 culture medium
Culture medium：Oat medium, 10% V8Culture medium, pumpkin culture medium, sponge gourd culture medium, lotus root culture medium, sweet potato training
Support base.
1.2 method
1.2.1 the preparation of culture medium
Pumpkin culture medium：20 ~ 40g pumpkins are cleaned into peeling, are cut into pieces, adds deionized water 50mL, uses tissue mashing machine
Smash about 40s to pieces, 2 layers of filtered through gauze remove slag, and supply water to 100mL, add in agar 2g, CaCO30.4g.The system of remaining culture medium
For with pumpkin culture medium.Each culture medium adjusts its pH before agar is added in, and the pH of culture medium is made to be maintained between 6.8 ~ 7.0.
121 DEG C of culture medium packing 250mL triangular flasks, the 20min prepared sterilizes, and treats that culture medium temperature is down to 50 DEG C of left sides
The right side, pours into culture medium in diameter 9cm sterilizing culture dishes on superclean bench, each culture dish 20mL culture mediums, and cooling is standby
With.
1.2.2 growth medium selects
Phytophthora nicotianae activates 2 ~ 3 times on oat medium tablet, 28 DEG C of dark culturings 7 ~ 10 days, with card punch by concentric
Annulus beats the mycelia block for taking diameter 5mm, and tweezers pipette ferfas silk face paste and are connected to each supply on examination culture medium flat plate.Each culture medium
5 tablets i.e. 5 repetitions, 28 DEG C of dark culturings.The the 3rd, 5 day measurement day growth speed is cultivated, records hyphae length（Mycelia thin and thick is dredged
It is close）, cover with the number of days of tablet.
1.2.3 spore ability is produced to measure
After examination culture medium inoculated culture 10 days, card punch, which is beaten, takes 15 pieces of diameter 5mm mycelia block in the empty culture dish of sterilizing,
Using mycelia clump water culture induction inducing spore capsule, alternating temperature release zoospore, count zoospore number.
2nd, result and analysis
The selection of 2.1 growth mediums
In 6 kinds of culture mediums for examination, all culture mediums can be grown, and colony colour is pure white, but mycelia gives birth to
Long speed there are significant difference, the speed of growth it is most fast be sweet potato culture medium, followed by pumpkin culture medium, oat medium, then
Secondary is sponge gourd culture medium, lotus root culture medium, and that worst is 10% V8Culture medium（Table 1）.In terms of the time for covering with tablet, 10% V8Training
Support base 3 days more late than other culture mediums, and other can cover with tablet in 6 days.
Growing state of 1 Phytophthora nicotianae of table on different culture media
2.2 production spore abilities measure
In 6 kinds of culture mediums, production spore capacity variance is notable, sponge gourd culture medium>Sweet potato culture medium>Lotus root culture medium>Pumpkin is trained
Support base>Oat medium>10% V8Culture medium, 10% V8There are the difference on the order of magnitude with other culture mediums for culture medium（Table 2）.
2 Phytophthora nicotianae of table production spore ability compares（10%V8 culture solutions induce）
2.3 Integrated comparative
In 6 kinds of culture mediums, Integrated comparative colony growth rate and production spore ability, it can be found that sweet potato culture medium, pumpkin
The culture medium either speed of growth and production spore ability are better than general oat medium, are more efficient substitutive mediums；
Although sponge gourd culture medium, the lotus root culture medium speed of growth are less than oat medium, production spore ability is significantly higher than oat medium, comprehensive
It closes efficiency and is better than oat medium.10%V8The medium culture time is longer, and overall efficiency is low, is not suitable for cultivating Phytophthora nicotianae Breda
Silk.
Claims (6)
1. a kind of Phytophthora nicotianae culture medium, it is characterised in that the Phytophthora nicotianae culture medium is with phytophagous substance, CaCO3With
Agar is prepared for raw material, in terms of 100mL, containing the juice 80 ~ 100mL, CaCO being prepared with phytophagous substance3 0.2~
1.7 ~ 2.5g of 0.4g and agar, is allocated using water as culture medium blender to rated capacity, the Phytophthora nicotianae Medium's PH Value
For 6.8 ~ 7.0；Phytophagous substance is pumpkin, sponge gourd, lotus root or sweet potato, and it is to take 20 ~ 40g phytophagous objects that phytophagous substance, which prepares juice,
Matter peeling section, adds in 40 ~ 80mL water, smashs to pieces, filter and remove residue, filtrate moisturizing to 100mL obtains.
2. the preparation method of Phytophthora nicotianae culture medium described in a kind of claim 1, it is characterised in that including pre-treatment, culture medium system
Standby, post-processing step, specifically includes：
A, pre-treatment：The peeling section of phytophagous substance is taken, phytophagous substance is pumpkin, sponge gourd, lotus root or sweet potato, adds in solid-liquid volume
Water than 2 ~ 5 times, is smashed to pieces, filter and remove residue, and filtrate moisturizing to rated capacity obtains phytophagous substance juice；
B, prepared by culture medium：Respectively phytophagous substance juice, CaCO are taken by formulation ratio3, it is 6.8 ~ 7.0 to adjust pH value, and addition is matched somebody with somebody
The agar just matched, heating make to obtain target culture medium after being completely dissolved；
C, post-process：Target culture medium is dispensed, sterilized processing finished product Phytophthora nicotianae culture medium.
3. preparation method according to claim 2, it is characterised in that smashing to pieces described in step A is smash using tissue mashing machine
Broken 30 ~ 50s.
4. preparation method according to claim 2, it is characterised in that the filter and remove residue described in step A is to take no less than 2
The hospital gauze of layer or special filtering filtration of material.
5. preparation method according to claim 2, it is characterised in that the sterilization treatment described in step C is to take damp and hot go out
One kind in bacterium, high-temperature sterilization or steam instantaneous sterilizing.
6. according to the preparation method of claim 2 or 5, it is characterised in that the sterilization treatment described in step C is to take 121 DEG C
20 ~ 40min of lower high-temperature sterilization.
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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 |
| 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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| CN102839148A (en) * | 2012-08-28 | 2012-12-26 | 云南农业大学 | Fluid medium made from celery and suitable for phytophthotacactorum to generate sporangium |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102839148A (en) * | 2012-08-28 | 2012-12-26 | 云南农业大学 | Fluid medium made from celery and suitable for phytophthotacactorum to generate sporangium |
| 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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