CN103436480B - Plate culture and preparation method of ustilaginoidea virens thin-wall conidium - Google Patents
Plate culture and preparation method of ustilaginoidea virens thin-wall conidium Download PDFInfo
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Abstract
The invention discloses a plate culture and preparation method of ustilaginoidea virens thin-wall conidium. The core of the method is that the thin-wall conidium is directly utilized for culturing and breeding new thin-wall conidium through a plate culture medium. The method comprises the following steps: 1) preparing an initial thin-wall conidium solution; 3) preparing a sporulation culture medium plate; 3) performing sporulation cultivation; 4) collecting new thin-wall conidium; and 5) purifying conidium. The method has the advantages that the cultivation and preparation can be performed within a shorter time which is generally 4 to 6 days; the operation is simple, easy and convenient; oscillating culture equipment is not required; the thin-wall conidium in an obtained culture product is mainly the conidium of the same generation; the conidium solution is relatively high in quality; no purifying treatment is required; and the method is applied to a plurality of research works.
Description
Technical field
The present invention relates to agrotechnique and biotechnology.The specifically conidial slat chain conveyor preparation method of a kind of ustilaginoidea virens thin-walled.
Background technology
In most of fungal disease system, the propagulum of pathogenic bacteria expands except the effects such as its population quantity except having breeding, the more important thing is to have and identify mutually with host, and start the function infecting host, therefore, in many important research of plant disease processes, all need to use the propagulum of pathogenic bacteria to make test materials, obviously, the cultivation preparation method of efficient propagulum material easily, will be very beneficial for carrying out smoothly of research work.False smut has become the great disease of one of China's Rice Production, and the propagulum of ustilaginoidea virens includes the thecaspore of condition, the pachypycnidium of Invisible element and thin-walled conidium three types.In laboratory, yet there are no so far by cultivating this germ and obtain thecasporous effective report; Cultivate acquisition pachypycnidium and also there is larger technical difficulty, and there is dormancy in pachypycnidium, uses very inconvenient; Therefore, the propagulum of relevant institute so far mainly uses the thin-walled conidium of this germ.
So far, the conidial cultivation preparation method of known ustilaginoidea virens thin-walled, mainly contains 2 kinds.One is the second stage of culture method, is namely first cultivated at solid medium by germ mycelium, and then mycelium is proceeded to liquid nutrient medium and carry out shaking culture, whole process generally needs more than 15 days.Another kind is three phase culture methods, namely first germ mycelium is cultivated at solid medium, then mycelium is proceeded to liquid nutrient medium and carry out shaking culture, then pour shaking culture product into solid medium again and carry out biphasic cultivation, whole process generally needs more than 20 days.These 2 kinds of methods have common step, all need to carry out agitated submerged culture.
These 2 kinds of methods effectively can prepare ustilaginoidea virens thin-walled conidium, but they all exist some disadvantages, as consuming time longer in practice; Work consuming is more; Need shaking culture equipment; Thin-walled conidium in cultured products belongs to the mixture of many spores from generation to generation; A large amount of hypha bodies, bacterial metabolism product and nutrient media components etc. are included in cultured products mixed solution, often need to filter the subsequent processing steps such as mycelia and precipitation spore to be just applicable to using, simultaneously because cultured products is viscous pasty state, precipitation spore often needs high speed centrifugation machine equipment.
Summary of the invention
The object of the invention is, according to after ustilaginoidea virens thin-walled conidia germination, there is special characteristic of growing, provide a kind of ustilaginoidea virens thin-walled conidial slat chain conveyor preparation method.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
The conidial slat chain conveyor preparation method of a kind of ustilaginoidea virens thin-walled, operation steps is as follows:
1. the preparation of initial thin-walled conidium liquid:
A) the initial conidial source of thin-walled: with known the second stage of cultural method, with mycelium by shaking culture mode, obtains containing the conidial cultured products of a large amount of thin-walled; Or adopt the fresh pachypycnidium cultivated on the sick grain of rice in field, obtain containing the conidial cultured products of a large amount of thin-walled.
B) spore purification process: conidial for the thin-walled of acquisition cultured products is carried out spore purification process, and purification treating method is, removes the mycelia in cultured products by 2 layers of filtered through gauze; Then filtered liquid is carried out centrifugal treating and collecting precipitation spore, the medium component in cultured products and the outer secretion matter of thalline can be removed; Then will precipitate spore suspension washing with sterilized water, then carry out a centrifugal treating, obtain pure spore ball.With sterilized water, namely spore ball suspended dispersed is evenly become thin-walled conidium liquid, save backup.
2. produce the preparation of spore culture medium flat plate:
Adopt half amount composition of PSA substratum as product spore substratum, producing spore substratum is: potato 100g; Sucrose 10g; Agar 20g; Water 1000ml, by changing into culture dish flat board after conventional sterilant, namely makes the product spore flat board producing spore and cultivate.
3. produce spore to cultivate:
Get step 1) the thin-walled conidium drop that saves backup is on the product spore culture medium flat plate face got ready, and by level and smooth glass stick trowelling platen surface, make that spore is full and uniform to be distributed in platen surface, proceeding to temperature after returning lid is cultivate 5 days under 28 DEG C of conditions.
4. the conidial collection of new thin-walled:
After upper step cultivates 5 days, product spore culture medium flat plate grows the conidial small colonies of generation thin-walled that a large amount of diameter is about 0.5 ~ 1.5mm, now collecting spore is the most in good time machine.Collection method is, gets clear water note in product spore platen surface, then, scrub with common hairbrush and produce spore culture medium flat plate surface colony, make spores release on bacterium colony in water, being collected by the water liquid scrubbing spore, is namely cultured products ustilaginoidea virens thin-walled conidium.
5. thin-walled conidium purification process:
Collected cultured products thin-walled conidium is carried out spore purification process, and purification treating method is, removes the mycelia in cultured products by 2 layers of filtered through gauze; Then filtered liquid is carried out centrifugal treating and collecting precipitation spore, the medium component in cultured products and the outer secretion matter of thalline can be removed; Then will precipitate spore suspension washing with sterilized water, then carry out a centrifugal treating, and very pure thin-walled can be obtained and divide spore ball.With sterilized water, namely this spore ball suspended dispersed is evenly become ustilaginoidea virens thin-walled point spore liquid.
Advantage of the present invention is:
1. cultivate preparation process time shorter, generally only need 4 ~ 6 days;
2. operation is simple and easy convenient, without the need to shaking culture equipment;
3. the spore of the mainly same generation of the thin-walled conidium in obtained cultured products;
4. spore liquid quality is higher, is also applicable to the use of many research work without the need to relevant purifying treatment.
Accompanying drawing explanation
Fig. 1 is the early stage schematic diagram of thin-walled conidia germination.
In figure, A is that spore starts to sprout generation germ tube; B is that the germ tube that spore germination produces constantly grows the sporogenous hyphae forming multi-branched; Black scale short-term 10 μm.
Fig. 2 is that the sporogenous hyphae of multi-branched progressively produces thin-walled conidium figure.
In figure, black scale line 0.4mm.
Fig. 3 is the continuous branch growth of sporogenous hyphae and produces spore, forms a mycelia and the intensive small colonies of spore is schemed.
In figure, mycelia and the spore of bacterium colony periphery are rarer, the good and imaging clearly (A) of printing opacity, the mycelia in the middle part of bacterium colony and spore intensive and light tight, cannot imaging (B).Black scale line: 0.6mm.
Fig. 4 is the bacterium colony figure that Fig. 3 amplifies 2 times.
In figure, A: to be on the good and camera lens focal plane of printing opacity more clearly spore string (group), B: be in printing opacity better and to depart from camera lens focal plane not clearly spore string (group); C: itself be in the light by thick close bacterium colony, the bacterium colony position that cannot focus.Black scale line: 0.4mm.
Fig. 5 is the partial enlarged drawing of Fig. 3 bacterium colony.
In figure, A: to be on the good ring district of colony edge printing opacity and camera lens focal plane more clearly spore string (group), B: be in printing opacity Shao Chahuan district and depart from camera lens focal plane, not clearly intensive spore string (group); C: be in mycelia and the thick close region of spore, light tight, imaging of cannot focusing.Black scale line: 0.2mm.
Fig. 6 is that ustilaginoidea virens forms a large amount of small colonies figure on product spore culture medium flat plate.
In figure, be applied in by thin-walled conidium to produce on spore substratum to cultivate after 5 days, each spore germination grows, and produces spore of new generation, forms the bacterium colony that diameter is about 0.5 ~ 1mm.
Fig. 7 is the mycelia figure of the bacterium colony periphery after product spore peak.
In figure, white proportion chi line: 0.4mm.
Fig. 8 is the partial enlargement of Fig. 7, shows that colony edge does not have sporulation in mycelia region clearly.
In figure, black scale line: 0.2mm.
Fig. 9 is that the inventive method cultivates the thin-walled conidium liquid figure obtained.
In figure, the outward appearance of the inventive method cultured products (A): liquid is evenly limpid, without obvious hypha body; Adopt the outward appearance of shaking culture method cultured products (B): liquid thickness, has a large amount of hypha body.
Embodiment
Below in conjunction with accompanying drawing and embodiment, the invention will be further described.
Growth and development process after ustilaginoidea virens thin-walled conidia germination is unique.Contriver finds, a thin-walled conidium sprouts on nutrient agar flat board, generally first produces germ tube, and germ tube produces the sporogenous hyphae (as shown in Figure 1) of multi-branched afterwards, on sporogenous hyphae, then form spore of new generation (as shown in Figure 2); Then, sporogenous hyphae is quick branch and large volume production spore more constantly, soon, sporogenous hyphae and spore set form macroscopic minute colony, the colony diameter produced when spore peaks is about 0.5 ~ 1.5mm, bacterium colony is now visible spore of new generation (as shown in Fig. 3 ~ Fig. 5) intensive in a large number under the microscope, and on culture medium flat plate, direct visual perception is small particles (as shown in Figure 6).After this, sporogenous hyphae grows changes the vegetative hyphae (as Suo Shi Fig. 7, Fig. 8) no longer producing spore into, and step by step, mycelia is increased, and bacterium colony increases, and spore amount then declines, until can't detect spore.The conidial slat chain conveyor preparation method of a kind of ustilaginoidea virens thin-walled of the present invention, utilizes the conidial this germination and growth characteristic of thin-walled exactly, collects in time at the initial stage of spore germination grown cultures, obtain thin-walled conidium new in a large number.
The conidial source of thin-walled of first use, 1) known the second stage of cultural method can be adopted, with mycelium by shaking culture mode, obtain containing the conidial cultured products of a large amount of thin-walled.2) also can adopt and of the present inventionly prepare spore method, cultivate the fresh pachypycnidium (chlamydospore) that field gathers, obtain containing the conidial cultured products of a large amount of thin-walled; This is easy to produce thin-walled conidium after sprouting based on pachypycnidium, and contriver finds, the characteristic of growing after pachypycnidium sprouting is the same with the characteristic of growing after thin-walled conidia germination.
Two kinds of methods in the conidial source of the thin-walled that above-mentioned explanation uses for the first time can obtain containing the conidial cultured products of thin-walled; After the spore purification process in this cultured products, can be stored in sterilized water for subsequent use for a long time; This finds based on contriver, deposits in sterilized water, preserve after 4 years and still have spore energy germination and growth to produce normal offspring, therefore, with sterilized water, simple thin-walled conidium can be made into simple thin-walled conidium spore liquid, save backup for a long time.
Embodiment 1
Adopt a kind of ustilaginoidea virens thin-walled of the present invention conidium to cultivate preparation method, cultivate the thin-walled conidium of preparing ustilaginoidea virens bacterial strain Uv-34, operate as follows:
1) preparation of initial thin-walled conidium liquid:
A) the conidial first source of thin-walled: adopt known the second stage of cultural method, with mycelium by shaking culture mode, obtains containing the conidial cultured products of a large amount of thin-walled;
B) spore purification process: carry out spore purification process by what obtain containing the conidial cultured products of thin-walled, purification treating method is, removes the mycelia in cultured products by 2 layers of filtered through gauze; Then filtered liquid is carried out centrifugal treating and collecting precipitation spore, the medium component in cultured products and the outer secretion matter of thalline can be removed; Then will precipitate spore suspension washing with sterilized water, then carry out a centrifugal treating, obtain pure spore ball.With sterilized water, spore ball suspended dispersed is even, and to be deployed into concentration be 10
6the spore liquid of individual spore/ml saves backup.
2) preparation of spore culture medium flat plate is produced:
Adopt half amount composition of PSA substratum as product spore substratum, producing spore medium component is: potato 100g; Sucrose 10g; Agar 20g; Water 1000ml, after conventional sterilant, pours 9cm culture dish into and makes the product spore flat board producing spore and cultivate.
3) produce spore to cultivate:
Get step 1) the thin-walled conidium liquid 10 μ l that saves backup, drip on the product spore culture medium flat plate face got ready, by level and smooth glass stick trowelling platen surface, make that spore is full and uniform to be distributed in platen surface, proceeding to temperature after returning lid is cultivate 5 days under 28 DEG C of conditions.
4) the conidial collection of new thin-walled:
After upper step cultivates 5 days, product spore culture medium flat plate grows generation thin-walled conidium small colonies (as shown in Figure 6) that a large amount of diameter is about 0.5 ~ 1.5mm, now for collecting new thin-walled the most in good time conidial machine.Collection method is, gets clear water note in product spore platen surface, then, scrub with common hairbrush and produce spore culture medium flat plate surface colony, make spores release on bacterium colony in water, each culture plate water 15ml scrubs 2 times, collects the cultured products of common acquisition 30ml; This cultured products contains thin-walled conidium new in a large number, and spore concentration reaches 1.89 × 10
6individual spore/ml.
Although this cultured products is also containing the outer secretion of a small amount of mycelia, nutrient media components and thalline, but compared with the Spore cultivation product obtained with shaking culture mode, spore liquid product appearance proterties of the present invention is evenly limpid, without significantly rolling into a ball saccharoid, move liquid or packaging operation good (as shown in Figure 9), thus spore liquid quality is higher, without relevant purification step, also the use of many research work is applicable to, very convenient.
5) thin-walled conidium purification process:
Collected cultured products is carried out thin-walled conidium purification process, purification treating method is, removes the mycelia in cultured products by 2 layers of filtered through gauze; Then filtered liquid is carried out centrifugal treating and collecting precipitation spore, the medium component in cultured products and the outer secretion matter of thalline can be removed; Then will precipitate spore suspension washing with sterilized water, then carry out a centrifugal treating, very pure thin-walled conidium group can be obtained; With sterilized water, namely this spore ball suspended dispersed is evenly become ustilaginoidea virens thin-walled conidium liquid.
Embodiment 2
A kind of ustilaginoidea virens thin-walled of the present invention conidium is adopted to cultivate preparation method, cultivate the thin-walled conidium of preparing ustilaginoidea virens bacterial strain Uv-105, implement to step 4) operation by the step 1) of embodiment 1, the every ware flat board of result obtains the cultured products 30ml containing thin-walled conidium liquid, and the spore concentration of cultured products reaches 2.72 × 10
6individual spore/ml; The appearance character of this cultured products and the identical of embodiment 1;
After implementing by the step 5) operation of embodiment 1, obtain the very pure thin-walled conidium of bacterial strain Uv-105.
Embodiment 3
A kind of ustilaginoidea virens thin-walled of the present invention conidium is adopted to cultivate preparation method, cultivate the thin-walled conidium of preparing ustilaginoidea virens bacterial strain Uv-110, implement to step 4) operation by the step 1) of embodiment 1, the every ware of result obtains the cultured products 30ml containing thin-walled conidium liquid, and the spore concentration of cultured products reaches 2.15 × 10
6individual spore/ml; The appearance character of this cultured products and the identical of embodiment 1;
After implementing by the step 5) operation of embodiment 1, obtain the very pure thin-walled conidium of bacterial strain Uv-110.
Embodiment 4
Adopt a kind of ustilaginoidea virens thin-walled of the present invention conidium to cultivate preparation method, cultivate the thin-walled conidium of preparing ustilaginoidea virens bacterial strain Uv-203, implementation and operation as follows:
1) preparation of initial thin-walled conidium liquid:
The conidial first source of thin-walled: the fresh pachypycnidium (chlamydospore) gathered from field, cultivates and obtains containing the conidial cultured products of a large amount of thin-walled; Concrete grammar is: gather the fresh rice curve in field (the sick grain of rice of false smut) and go back to laboratory; Aseptically, clamp rice curve with tweezers, knock tweezers gently, shake and abandon the pachypycnidium on rice curve top layer, then rice curve is moved on to and produce above spore substratum, then knock tweezers, the pachypycnidium on rice curve is shaken to fall to interspersing among and cultivates on basal plane, cover culture dish lid, being placed in temperature is cultivate under the condition of 28 DEG C, after 5 days, cultivates on basal plane and occurs that many diameters are the white small colonies of 0.5 ~ 1mm, meanwhile, also there is some contaminants or bacterial colony; By sterile razor blade, free of contamination ustilaginoidea virens small colonies is transferred in sterile petri dish together with culture medium flat plate cutting, add a small amount of sterilized water, scrub small colonies gently with sterilizing hairbrush, obtain containing the conidial initial spore liquid of a large amount of thin-walled; This spore liquid can adopt method of the present invention without purification process, be directly used for cultivating the new thin-walled conidium of preparation; Ensuing operation steps is as follows:
2) preparation of spore culture medium flat plate is produced: the step 2 by embodiment 1) operation enforcement;
3) produce spore to cultivate: get the present embodiment step 1) cultivate the thin-walled conidium liquid 10 μ l obtained, drip on the product spore culture medium flat plate face got ready, by level and smooth glass stick trowelling platen surface, make that spore is full and uniform to be distributed in platen surface, proceeding to temperature after returning lid is cultivate under 28 DEG C of conditions.
4) the conidial collection of new thin-walled: implement by the step 4) operation of embodiment 1; The every ware flat board of result obtains the cultured products 30ml containing thin-walled conidium liquid, and the spore concentration of cultured products reaches 2.52 × 10
6individual spore/ml; The appearance character of this cultured products and the identical of embodiment 1;
5) thin-walled conidium purification process: after implementing by the step 5) operation of embodiment 1, obtain very pure thin-walled conidium; This bacterial strain is directly obtain from the Pathogen culture the sick grain of rice in field, names as Uv-203.
Embodiment 5
Adopt a kind of ustilaginoidea virens thin-walled of the present invention conidium to cultivate preparation method, cultivate the thin-walled conidium of preparing ustilaginoidea virens bacterial strain Uv-205.Implement to step 4) operation by the step 1) of embodiment 4, the every ware of result obtains the cultured products 30ml containing thin-walled conidium liquid, and the spore concentration of cultured products is 1.25 × 10
6individual spore/ml; The appearance character of this cultured products and the identical of embodiment 1;
After implementing by the step 5) operation of embodiment 1, obtain very pure thin-walled conidium; This bacterial strain is directly obtain from the Pathogen culture the sick grain of rice in field, names as Uv-205.
Claims (1)
1. the conidial slat chain conveyor preparation method of ustilaginoidea virens thin-walled, it is characterized in that, operation steps is as follows:
1) preparation of initial thin-walled conidium liquid:
A) the initial conidial source of thin-walled: adopt known the second stage of cultural method, with mycelium by shaking culture mode, obtains containing the conidial cultured products of a large amount of thin-walled; Or adopt the method for the fresh pachypycnidium cultivated on the sick grain of rice in field, obtain containing the conidial cultured products of a large amount of thin-walled;
B) spore purification process: conidial for the thin-walled of acquisition cultured products is carried out spore purification process, and purification treating method is, removes the mycelia in cultured products by 2 layers of filtered through gauze; Then filtered liquid is carried out centrifugal and collecting precipitation spore; Then will precipitate spore suspension washing with sterilized water, then carry out once centrifugal, and obtain pure spore ball, with sterilized water, namely spore ball suspended dispersed is evenly become thin-walled conidium liquid, save backup;
2) preparation of spore culture medium flat plate is produced:
Adopt half amount composition of PSA substratum as product spore substratum, producing spore substratum is: potato 100g; Sucrose 10g; Agar 20g; Water 1000ml, by changing into culture dish flat board after conventional sterilant, namely makes the product spore flat board producing spore and cultivate;
3) produce spore to cultivate:
Get step 1) the thin-walled conidium liquid that saves backup, drip on the product spore culture medium flat plate face got ready, by level and smooth glass stick trowelling platen surface, make that spore is full and uniform to be distributed in platen surface, proceeding to temperature after returning lid is cultivate 5 days under 28 DEG C of conditions;
4) the conidial collection of new thin-walled:
After upper step cultivates 5 days, product spore culture medium flat plate grows the conidial small colonies of generation thin-walled that a large amount of diameter is about 0.5 ~ 1.5mm, now collect new thin-walled conidium; Collection method is, get clear water note in product spore platen surface, then scrub with common hairbrush and produce spore culture medium flat plate surface colony, make spores release on bacterium colony in water, the water liquid scrubbing spore is collected, is containing the conidial cultured products of ustilaginoidea virens thin-walled new in a large number;
5) thin-walled conidium purification process:
Collected cultured products is carried out thin-walled conidium purification process, purification treating method is, removes the mycelia in cultured products by 2 layers of filtered through gauze; Then filtered liquid is carried out centrifugal treating and collecting precipitation spore; Then will precipitate spore suspension washing with sterilized water, then carry out a centrifugal treating, and very pure thin-walled conidium group can be obtained, with sterilized water, namely this spore ball suspended dispersed is evenly become ustilaginoidea virens thin-walled conidium liquid.
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| CN105779372A (en) * | 2016-05-24 | 2016-07-20 | 广西大学 | Potato culture medium suitable for ustilaginoidea virens spore production and application thereof |
| CN106085945B (en) * | 2016-08-24 | 2020-08-18 | 文山苗乡三七科技有限公司 | Method for inducing conidia of panax notoginseng orbicularis |
| CN106701653A (en) * | 2017-01-10 | 2017-05-24 | 广西大学 | Pollution-reduction preparation and culture method of ustilaginoidea virens thin-wall conidia |
| CN106635948A (en) * | 2017-01-10 | 2017-05-10 | 广西大学 | Pollution-free preparing and culturing method for rice-false-smut-case thin-wall conidia |
| CN106635841A (en) * | 2017-01-10 | 2017-05-10 | 广西大学 | Standing form of botrytis cinerea laboratory material and preparation and application method of standing form |
| CN106867957A (en) * | 2017-01-10 | 2017-06-20 | 广西大学 | A kind of ustilaginoidea virens thin-walled of Pollution protection is conidial to prepare cultural method |
| CN113308432B (en) * | 2021-06-15 | 2022-09-16 | 广西大学 | Transfer and dispersion method of ustilaginoidea virens thin-walled conidia |
| CN113308430B (en) * | 2021-06-15 | 2022-09-16 | 广西大学 | Dispersion culture method of banana colletotrichum gloeosporioides conidia |
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