CN102559516A - Tobacco black shank pathogen liquid-solid composite system culturing method and application of cultures - Google Patents
Tobacco black shank pathogen liquid-solid composite system culturing method and application of cultures Download PDFInfo
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Abstract
The invention discloses a tobacco black shank pathogen liquid-solid composite system culturing method and an application of cultures. Solids obtained by culturing through the method can be applied to artificial inoculums of the tobacco black shank and can also be used for preparing infected soil additives with tobacco black shank pathogens to conveniently determine the resistance of the black shank by the kind of tobaccos and screen medicines, biocontrol bacteria or natural products for controlling the tobacco black shank. The culturing method comprises the following steps: 1, carrying out pathogen separation: acquiring infected strains from an infected field, separating, purifying, and determining the pathogenicity to obtain the pathogens; 2, carrying out plat culturing: inoculating the pathogens onto a medium flat, and culturing for 5-7 days at 24-30DEG C; 3, carrying out liquid culturing: inoculating flat mycelia into a culturing solution, and oscillate-culturing for 5-7 days under conditions that the temperature is 24-30DEG C and the oscillation speed is 150p/min; and 4, carrying out solid culturing: inoculating liquid mycelia into a solid medium, and culturing for 8-12 days at 24-30DEG C. The method has the advantages of large inoculation amount, short culturing period, difficult pollution and the like.
Description
Technical field
The invention belongs to agriculture microbial fermentation engineering technical field, be specifically related to the application of a kind of black shank germ liquid-solid composite system cultural method and culture.
Background technology
Black shank (tobacco black shank) is to endanger one of severe diseases in the World tobacco production, particularly in the temperate zone, subtropics and the torrid zone take place heavylier, also are the main disease types that China tobacco produces.At present, in the laboratory, the greenhouse has and adopts dull and stereotyped mycelia piece, blends the method that mycelia liquid, zoospore liquid etc. prepare black shank germ inoculum.Inoculum complicated operation, the technical difficulty of these several class methods preparations are big, generally are not suitable for the land for growing field crops artificial inoculation.
What the application of black shank germ inoculum was more in the field is that cereal is cultivated inoculum, and the artificial inoculation pathogen is used when mainly supplying evaluation of tobacco germplasm resistance or medicament screening.This cereal cultivate be with the mycelia piece direct inoculation on the flat board to the cereal media surface, let mycelia by around the introversion, grow from top to bottom, spread.Owing to be the inoculation of single-point or some top layer, mycelia needs 45 ~ 60 talentes possibly cover with whole matrix usually, and the cycle is longer; The levels mycelial growth is asynchronous during cultivation, causes the top mycelia excessively aging and the bottom mycelia is too young tender, and the solid culture heterogeneity influences infecting of germ, causes very big personal errors; And the germ mycelia can not capture matrix very soon and is the chance that other assorted bacterium that possibly pollute provide growth when cultivating, and the living contaminants that causes germ to be cultivated is cultivated the germ failure.In addition, the grain of cereal cultivation inoculum is excessive, and excessive nutrition cause antagonistic microbe to breed rapidly, and effective quantity of germ inoculum sharply descends for its potential antagonistic microbe provides abundant nutriment, can not form effective dip-dye.Therefore, how effectively solid culture black shank germ is the technical problem that is worth exploration in tobacco breeding, the plant protection work.
Summary of the invention
Below in conjunction with embodiment the present invention is further described, but never in any form the present invention is limited, any change or improvement based on training centre of the present invention is done all belong to protection scope of the present invention.
First purpose of the present invention realizes through following technical scheme, comprises germ separation, dull and stereotyped cultivation, liquid culture, solid culture, specifically may further comprise the steps:
A, germ separation are gathered diseased plant from the piece of morbidity field, measure through separation, purifying, virulence, obtain germ, adopt mycelia piece water seaoning to place constant temperature preservation under 10 ℃ of conditions bacterial classification, and be subsequent use;
B, the dull and stereotyped cultivation are inoculated in germ on the culture medium flat plate, under 24 ~ 30 ℃ of temperature, cultivate 5 ~ 7 days, select the mycelia pure white, at the culture of the densely covered one deck mycelia of planar surface, as the liquid culture bacterial classification;
C, liquid culture; The dull and stereotyped mycelium inoculation that the B step is obtained is in nutrient solution; Under 24 ~ 30 ℃ of temperature, shaking culture is 5 ~ 7 days under 130 ~ 150r/min rotating speed, cultivates white mycelium in the liquid bacterium liquid obtain, bacterium liquid is not muddy, mycelia is not agglomerating; Microscopy can be found zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 10,000;
D, solid culture; The liquid mycelium inoculation that the C step is obtained is in solid medium; Cultivated 8 ~ 12 days for 24 ~ 30 ℃, cultivate the whole matrix of solid culture that obtains and be covered with white hypha, mycelia discharges spore through soaked with liquid, low temperature induction, room temperature; Microscopy visible zoosporangium and zoospore or the spore that stops, every milliliter of spore content is more than 100,000.
Another object of the present invention is achieved in that culture is used for the artificial inoculation thing of black shank, perhaps is used to prepare the additive of band black shank germ sick soil.
The present invention has the following advantages and effect with respect to prior art:
(1) training quality is stable.The present invention can make germ capture matrix rapidly through increasing the inoculum size that the liquid culture step increases solid culture, reaches synchronously, grows fast; Growth cycle is short; Than conventional incubation time 10 ~ 15 days of shortening, under the prerequisite of Strict aseptic operation, can stop living contaminants; Through each flow process of strict control cultivation, thus the complete quality assurance measure of formation science; And each flow process time span of control is narrow, can rationally arrange incubation time, ensures the ageing of germ training quality;
(2) cultivate germ effectively to infect quantity big.It is littler 3 ~ 5 times than the grain of common usefulness to cultivate used particle; Germ can occupy whole grain matrix, and superfluous nutritive substance is few, can promote the continuation breeding of germ like this; Can avoid again potential antagonistic microbe growth, make germ avoid clearing up, germ effectively to infect quantity big.
Embodiment
Below in conjunction with embodiment the present invention is further described, but never in any form the present invention is limited, any change or improvement based on training centre of the present invention is done all belong to protection scope of the present invention.
Comprise germ separation, dull and stereotyped cultivation, liquid culture, solid culture, specifically may further comprise the steps:
A, germ separation are gathered diseased plant from the piece of morbidity field, measure through separation, purifying, virulence, obtain germ, adopt mycelia piece water seaoning to place constant temperature preservation under 10 ℃ of conditions bacterial classification, and be subsequent use;
B, the dull and stereotyped cultivation are inoculated in germ on the culture medium flat plate, under 24 ~ 30 ℃ of temperature, cultivate 5 ~ 7 days, select the mycelia pure white, at the culture of the densely covered one deck mycelia of planar surface, as the liquid culture bacterial classification;
C, liquid culture; The dull and stereotyped mycelium inoculation that the B step is obtained is in nutrient solution; Under 24 ~ 30 ℃ of temperature, shaking culture is 5 ~ 7 days under 130 ~ 150r/min rotating speed, cultivates white mycelium in the liquid bacterium liquid obtain, bacterium liquid is not muddy, mycelia is not agglomerating; Microscopy can be found zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 10,000;
D, solid culture; The liquid mycelium inoculation that the C step is obtained is in solid medium; Cultivated 8 ~ 12 days for 24 ~ 30 ℃, cultivate the whole matrix of solid culture that obtains and be covered with white hypha, mycelia discharges spore through soaked with liquid, low temperature induction, room temperature; Microscopy visible zoosporangium and zoospore or the spore that stops, every milliliter of spore content is more than 100,000.
Described B step substratum is any one in oat, sesame, rye, Semen Phaseoli Vulgaris, lima bean, the soya broth.
In the oat that described C step nutrient solution is 20 ~ 30g, sesame, rye, Semen Phaseoli Vulgaris, lima bean, the soybean any one; After soaking 18 ~ 28 hours in the adding 1000mL zero(ppm) water; Smash tissue to pieces after-filtration; Conventional damp and hot method sterilization, add with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 10 ~ 20g is smashed to pieces in a manner described, filtration sterilization prepares cigarette root tissue liquid mixing again, said nutrient solution accounts for 30% ~ 50% of vessel volume.
Described C step nutrient solution is any one in 150 ~ 250g fresh carrot, tomato, big bean sprouts, the Semen Phaseoli radiati Germinatus, adds 1000mL and heats up in a steamer water, smashs tissue to pieces after-filtration, more successively through sterilizing behind 0.45 μ m, the 0.22 μ m membrane filtration; Add with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 10 ~ 20g is smashed to pieces in a manner described, filtration sterilization prepares cigarette root tissue liquid mixing again, said nutrient solution accounts for 30% ~ 50% of vessel volume.
The dull and stereotyped mycelium inoculation of described C step is to cut black shank germ mycelia piece with the scalper of sterilizing in nutrient solution, and the mycelia piece size is 2 ~ 4 * 2 ~ 4mm, and the solid medium carrier thickness is 1 ~ 2mm, 2 ~ 4 of per 100 milliliters of nutrient solutions inoculation mycelia.
In the brown shorts that described D step solid medium is particle diameter 1 ~ 3mm or pulverizing pea bar or the broad bean bar one or more; Compare mixing with brown shorts with the quality of 3 ~ 4:1 ~ 2; The abundant mixing of water of adding 40% ~ 50%, 50% ~ 80% of solid culture fiduciary point vessel volume, conventional moist heat sterilization.
Than mixing, adding water mixture control water cut is 40% ~ 50%, 50% ~ 80% of solid culture fiduciary point vessel volume, conventional moist heat sterilization with the quality of 6 ~ 8:1 ~ 2 for fresh residue from beans after making that described D step solid medium is particle diameter 1 ~ 3mm and brown shorts.
Described D step liquid mycelium inoculation adds by 3% ~ 5% volume ratio in the solid culture base system, adds the no hydrops in substratum bottom, back.
Embodiment 1
To from the piece of morbidity field, gather diseased plant, measure, obtain germ, and adopt mycelia piece water seaoning to place constant temperature preservation under 10 ℃ of conditions bacterial classification through separation, purifying, virulence, subsequent use; It is the 10g oat is soaked 24 hours in 1000mL zero(ppm) water after, to smash tissue to pieces after-filtration that germ is inoculated in substratum, conventional damp and hot method sterilization; Add cigarette root tissue liquid mixing again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 10g is smashed to pieces in a manner described, filtration sterilization prepares; And process plate, and under 24 ℃ of temperature, cultivated 5 days, select the mycelia pure white; At the culture of the densely covered one deck mycelia of planar surface, as the liquid culture bacterial classification; The dull and stereotyped mycelium inoculation that will obtain is again smashed tissue to pieces after-filtration in heating up in a steamer water with 150g fresh carrot adding 1000mL, more successively through sterilizing behind 0.45 μ m, the 0.22 μ m membrane filtration; Add again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 10g is smashed to pieces in a manner described, filtration sterilization prepares cigarette root tissue liquid mixing in the nutrient solution; Dull and stereotyped mycelium inoculation is to cut black shank germ mycelia piece with the sterilization scalper in nutrient solution; The mycelia piece size is 4 * 4mm; The solid medium carrier thickness is 1.9mm, 2 of per 100 milliliters of nutrient solutions inoculation mycelia.The inoculation back is under 24 ℃ of temperature; Shaking culture is 7 days under the 130r/min rotating speed; White mycelium in the liquid bacterium liquid that cultivation obtains, bacterium liquid is not muddy, mycelia is not agglomerating, and microscopy can be found zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 10,000; The liquid mycelium inoculation that obtains is compared mixing in the fresh residue from beans after making of particle diameter 1mm and brown shorts with the quality of 8:2; Controlling moisture content is in 45% solid medium; The volume ratio that the liquid mycelia is pressed solid medium 4% adds, and adds the no hydrops in substratum bottom, back, cultivates 9 days for 28 ℃; The whole matrix of the solid culture that cultivation obtains is covered with white hypha; Mycelia discharges spore through soaked with liquid, low temperature induction, room temperature, microscopy visible zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 100,000.The culture that obtains can be used as the artificial inoculation thing of black shank, perhaps is used to prepare the usefulness of the additive of being with black shank germ sick soil.
Embodiment 2
To from the piece of morbidity field, gather diseased plant, measure, obtain germ, and adopt mycelia piece water seaoning to place constant temperature preservation under 10 ℃ of conditions bacterial classification through separation, purifying, virulence, subsequent use; It is the 30g sesame soaks 18 hours in 1000mL zero(ppm) water after, to smash tissue to pieces after-filtration that germ is inoculated in substratum, conventional damp and hot method sterilization; Add cigarette root tissue liquid mixing again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 20g is smashed to pieces in a manner described, filtration sterilization prepares; And process plate, and under 30 ℃ of temperature, cultivated 5 days, select the mycelia pure white; At the culture of the densely covered one deck mycelia of planar surface, as the liquid culture bacterial classification; The dull and stereotyped mycelium inoculation that will obtain is again smashed tissue to pieces after-filtration in heating up in a steamer water with 150g fresh carrot adding 1000mL, more successively through sterilizing behind 0.45 μ m, the 0.22 μ m membrane filtration; Add again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 10g is smashed to pieces in a manner described, filtration sterilization prepares cigarette root tissue liquid mixing in the nutrient solution; Dull and stereotyped mycelium inoculation is to cut black shank germ mycelia piece with the sterilization scalper in nutrient solution; The mycelia piece size is 2 * 2mm; The solid medium carrier thickness is 1mm, 2 of per 100 milliliters of nutrient solutions inoculation mycelia.The inoculation back is under 30 ℃ of temperature; Shaking culture is 7 days under the 150r/min rotating speed; White mycelium in the liquid bacterium liquid that cultivation obtains, bacterium liquid is not muddy, mycelia is not agglomerating, and microscopy can be found zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 10,000; The liquid mycelium inoculation that obtains is compared mixing in brown shorts and brown shorts with the quality of 4:1; The solid medium that the abundant mixing of water of adding 50% makes; The volume ratio that the liquid mycelia is pressed solid medium 4.5% adds, and adds the no hydrops in substratum bottom, back, cultivates 10 days for 24 ℃; The whole matrix of the solid culture that cultivation obtains is covered with white hypha; Mycelia discharges spore through soaked with liquid, low temperature induction, room temperature, microscopy visible zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 100,000.The culture that obtains can be used as the artificial inoculation thing of black shank, perhaps is used to prepare the usefulness of the additive of being with black shank germ sick soil.
Embodiment 3
To from the piece of morbidity field, gather diseased plant, measure, obtain germ, and adopt mycelia piece water seaoning to place constant temperature preservation under 10 ℃ of conditions bacterial classification through separation, purifying, virulence, subsequent use; It is the 28g lima bean soaks 28 hours in 1000mL zero(ppm) water after, to smash tissue to pieces after-filtration that germ is inoculated in substratum, conventional damp and hot method sterilization; Add cigarette root tissue liquid mixing again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 15g is smashed to pieces in a manner described, filtration sterilization prepares; And process plate, and under 28 ℃ of temperature, cultivated 6 days, select the mycelia pure white; At the culture of the densely covered one deck mycelia of planar surface, as the liquid culture bacterial classification; The dull and stereotyped mycelium inoculation that will obtain is again smashed tissue to pieces after-filtration in heating up in a steamer water with 250g fresh tomato adding 1000mL, more successively through sterilizing behind 0.45 μ m, the 0.22 μ m membrane filtration; Add again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 15g is smashed to pieces in a manner described, filtration sterilization prepares cigarette root tissue liquid mixing in the nutrient solution; Dull and stereotyped mycelium inoculation is to cut black shank germ mycelia piece with the sterilization scalper in nutrient solution; The mycelia piece size is 4 * 4mm; The solid medium carrier thickness is 2mm, 4 of per 100 milliliters of nutrient solutions inoculation mycelia.The inoculation back is under 28 ℃ of temperature; Shaking culture is 6 days under the 140r/min rotating speed; White mycelium in the liquid bacterium liquid that cultivation obtains, bacterium liquid is not muddy, mycelia is not agglomerating, and microscopy can be found zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 10,000; The liquid mycelium inoculation that obtains is compared mixing in pea bean straw powder and brown shorts with the quality of 3:1; The solid medium that the abundant mixing of water of adding 45% makes; The volume ratio that the liquid mycelia is pressed solid medium 3.5% adds, and adds the no hydrops in substratum bottom, back, cultivates 9 days for 28 ℃; The whole matrix of the solid culture that cultivation obtains is covered with white hypha; Mycelia discharges spore through soaked with liquid, low temperature induction, room temperature, microscopy visible zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 100,000.The culture that obtains can be used as the artificial inoculation thing of black shank, perhaps is used to prepare the usefulness of the additive of being with black shank germ sick soil.
Embodiment 4
To from the piece of morbidity field, gather diseased plant, measure, obtain germ, and adopt mycelia piece water seaoning to place constant temperature preservation under 10 ℃ of conditions bacterial classification through separation, purifying, virulence, subsequent use; It is the 20g Semen Phaseoli Vulgaris soaks 28 hours in 1000mL zero(ppm) water after, to smash tissue to pieces after-filtration that germ is inoculated in substratum, conventional damp and hot method sterilization; Add cigarette root tissue liquid mixing again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 10g is smashed to pieces in a manner described, filtration sterilization prepares; And process plate, and under 24 ℃ of temperature, cultivated 7 days, select the mycelia pure white; At the culture of the densely covered one deck mycelia of planar surface, as the liquid culture bacterial classification; The dull and stereotyped mycelium inoculation that will obtain is again smashed tissue to pieces after-filtration in heating up in a steamer water with 150g fresh soyabean bud adding 1000mL, more successively through sterilizing behind 0.45 μ m, the 0.22 μ m membrane filtration; Add again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 20g is smashed to pieces in a manner described, filtration sterilization prepares cigarette root tissue liquid mixing in the nutrient solution; Dull and stereotyped mycelium inoculation is to cut black shank germ mycelia piece with the sterilization scalper in nutrient solution; The mycelia piece size is 3 * 3mm; The solid medium carrier thickness is 1.5mm, 3 of per 100 milliliters of nutrient solutions inoculation mycelia.The inoculation back is under 24 ℃ of temperature; Shaking culture is 7 days under the 130r/min rotating speed; White mycelium in the liquid bacterium liquid that cultivation obtains, bacterium liquid is not muddy, mycelia is not agglomerating, and microscopy can be found zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 10,000; The liquid mycelium inoculation that obtains is compared mixing in silkworm bean straw powder and brown shorts with the quality of 3:1.5; The solid medium that the abundant mixing of water of adding 50% makes; The volume ratio that the liquid mycelia is pressed solid medium 3% adds, and adds the no hydrops in substratum bottom, back, cultivates 10 days for 27 ℃; The whole matrix of the solid culture that cultivation obtains is covered with white hypha; Mycelia discharges spore through soaked with liquid, low temperature induction, room temperature, microscopy visible zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 100,000.The culture that obtains can be used as the artificial inoculation thing of black shank, perhaps is used to prepare the usefulness of the additive of being with black shank germ sick soil.
Embodiment 5
To from the piece of morbidity field, gather diseased plant, measure, obtain germ, and adopt mycelia piece water seaoning to place constant temperature preservation under 10 ℃ of conditions bacterial classification through separation, purifying, virulence, subsequent use; It is the 25g rye is soaked 23 hours in 1000mL zero(ppm) water after, to smash tissue to pieces after-filtration that germ is inoculated in substratum, conventional damp and hot method sterilization; Add cigarette root tissue liquid mixing again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 14g is smashed to pieces in a manner described, filtration sterilization prepares; And process plate, and under 29 ℃ of temperature, cultivated 6 days, select the mycelia pure white; At the culture of the densely covered one deck mycelia of planar surface, as the liquid culture bacterial classification; The dull and stereotyped mycelium inoculation that will obtain is again smashed tissue to pieces after-filtration in heating up in a steamer water with the fresh Semen Phaseoli radiati Germinatus adding of 250g 1000mL, more successively through sterilizing behind 0.45 μ m, the 0.22 μ m membrane filtration; Add again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 20g is smashed to pieces in a manner described, filtration sterilization prepares cigarette root tissue liquid mixing in the nutrient solution; Dull and stereotyped mycelium inoculation is to cut black shank germ mycelia piece with the sterilization scalper in nutrient solution; The mycelia piece size is 4 * 4mm; The solid medium carrier thickness is 2mm, 2 of per 100 milliliters of nutrient solutions inoculation mycelia.The inoculation back is under 25 ℃ of temperature; Shaking culture is 6 days under the 150r/min rotating speed; White mycelium in the liquid bacterium liquid that cultivation obtains, bacterium liquid is not muddy, mycelia is not agglomerating, and microscopy can be found zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 10,000; With the liquid mycelium inoculation that obtains in brown shorts with after pea bean straw powder 1:1 mixes; Again with brown shorts with the quality of 3:2 than mixing, add the solid medium that 50% the abundant mixing of water makes, the liquid mycelia press the volume ratio adding of solid medium 4%; Add the no hydrops in substratum bottom, back; Cultivated 8 days for 29 ℃, cultivate the whole matrix of solid culture that obtains and be covered with white hypha, mycelia discharges spore through soaked with liquid, low temperature induction, room temperature; Microscopy visible zoosporangium and zoospore or the spore that stops, every milliliter of spore content is more than 100,000.The culture that obtains can be used as the artificial inoculation thing of black shank, perhaps is used to prepare the usefulness of the additive of being with black shank germ sick soil.
Embodiment 6
To from the piece of morbidity field, gather diseased plant, measure, obtain germ, and adopt mycelia piece water seaoning to place constant temperature preservation under 10 ℃ of conditions bacterial classification through separation, purifying, virulence, subsequent use; It is the 20g soybean is soaked 27 hours in 1000mL zero(ppm) water after, to smash tissue to pieces after-filtration that germ is inoculated in substratum, conventional damp and hot method sterilization; Add cigarette root tissue liquid mixing again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 17g is smashed to pieces in a manner described, filtration sterilization prepares; And process plate, and under 30 ℃ of temperature, cultivated 5 days, select the mycelia pure white; At the culture of the densely covered one deck mycelia of planar surface, as the liquid culture bacterial classification; The dull and stereotyped mycelium inoculation that will obtain is again smashed tissue to pieces after-filtration in heating up in a steamer water with 150g fresh carrot and 100g tomato adding 1000mL, more successively through sterilizing behind 0.45 μ m, the 0.22 μ m membrane filtration; Add again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 10g is smashed to pieces in a manner described, filtration sterilization prepares cigarette root tissue liquid mixing in the nutrient solution; Dull and stereotyped mycelium inoculation is to cut black shank germ mycelia piece with the sterilization scalper in nutrient solution; The mycelia piece size is 2.5 * 2.5mm; The solid medium carrier thickness is 1.7mm, 3 of per 100 milliliters of nutrient solutions inoculation mycelia.The inoculation back is under 28 ℃ of temperature; Shaking culture is 5 days under the 140r/min rotating speed; White mycelium in the liquid bacterium liquid that cultivation obtains, bacterium liquid is not muddy, mycelia is not agglomerating, and microscopy can be found zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 10,000; The liquid mycelium inoculation that obtains is compared mixing in the mixture of the brown shorts of particle diameter 1mm and silkworm bean straw powder and brown shorts with the quality of 4:1; The solid medium that the abundant mixing of water of adding 50% makes; The volume ratio that the liquid mycelia is pressed solid medium 5% adds, and adds the no hydrops in substratum bottom, back, cultivates 10 days for 24 ℃; The whole matrix of the solid culture that cultivation obtains is covered with white hypha; Mycelia discharges spore through soaked with liquid, low temperature induction, room temperature, microscopy visible zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 100,000.The culture that obtains can be used as the artificial inoculation thing of black shank, perhaps is used to prepare the usefulness of the additive of being with black shank germ sick soil.
Embodiment 7
To from the piece of morbidity field, gather diseased plant, measure, obtain germ, and adopt mycelia piece water seaoning to place constant temperature preservation under 10 ℃ of conditions bacterial classification through separation, purifying, virulence, subsequent use; It is the 20g oat is soaked 26 hours in 1000mL zero(ppm) water after, to smash tissue to pieces after-filtration that germ is inoculated in substratum, conventional damp and hot method sterilization; Add cigarette root tissue liquid mixing again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 18g is smashed to pieces in a manner described, filtration sterilization prepares; And process plate, and under 30 ℃ of temperature, cultivated 5 days, select the mycelia pure white; At the culture of the densely covered one deck mycelia of planar surface, as the liquid culture bacterial classification; The dull and stereotyped mycelium inoculation that will obtain is again smashed tissue to pieces after-filtration in heating up in a steamer water with 130g fresh carrot and 120g tomato adding 1000mL, more successively through sterilizing behind 0.45 μ m, the 0.22 μ m membrane filtration; Add again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 17g is smashed to pieces in a manner described, filtration sterilization prepares cigarette root tissue liquid mixing in the nutrient solution; Dull and stereotyped mycelium inoculation is to cut black shank germ mycelia piece with the sterilization scalper in nutrient solution; The mycelia piece size is 3.5 * 3.5mm; The solid medium carrier thickness is 1.9mm, 4 of per 100 milliliters of nutrient solutions inoculation mycelia.The inoculation back is under 24 ℃ of temperature; Shaking culture is 7 days under the 130r/min rotating speed; White mycelium in the liquid bacterium liquid that cultivation obtains, bacterium liquid is not muddy, mycelia is not agglomerating, and microscopy can be found zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 10,000; The liquid mycelium inoculation that obtains is compared mixing in the fresh residue from beans after making of particle diameter 3mm and brown shorts with the quality of 6:1; Controlling moisture content is in 45% solid medium; The volume ratio that the liquid mycelia is pressed solid medium 3% adds, and adds the no hydrops in substratum bottom, back, cultivates 9 days for 28 ℃; The whole matrix of the solid culture that cultivation obtains is covered with white hypha; Mycelia discharges spore through soaked with liquid, low temperature induction, room temperature, microscopy visible zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 100,000.The culture that obtains can be used as the artificial inoculation thing of black shank, perhaps is used to prepare the usefulness of the additive of being with black shank germ sick soil.
Claims (9)
1. the liquid-solid composite system cultural method of a black shank germ comprises germ separation, dull and stereotyped cultivation, liquid culture, solid culture, it is characterized in that cultural method specifically may further comprise the steps:
A, germ separation are gathered diseased plant from the piece of morbidity field, measure through separation, purifying, virulence, obtain germ, adopt mycelia piece water seaoning to place constant temperature preservation under 10 ℃ of conditions bacterial classification, and be subsequent use;
B, the dull and stereotyped cultivation are inoculated in germ on the culture medium flat plate, under 24 ~ 30 ℃ of temperature, cultivate 5 ~ 7 days, select the mycelia pure white, at the culture of the densely covered one deck mycelia of planar surface, as the liquid culture bacterial classification;
C, liquid culture; The dull and stereotyped mycelium inoculation that the B step is obtained is in nutrient solution; Under 24 ~ 30 ℃ of temperature, shaking culture is 5 ~ 7 days under 130 ~ 150r/min rotating speed, cultivates white mycelium in the liquid bacterium liquid obtain, bacterium liquid is not muddy, mycelia is not agglomerating; Microscopy can be found zoosporangium and zoospore or the spore that stops, and every milliliter of spore content is more than 10,000;
D, solid culture; The liquid mycelium inoculation that the C step is obtained is in solid medium; Cultivated 8 ~ 12 days for 24 ~ 30 ℃, cultivate the whole matrix of solid culture that obtains and be covered with white hypha, mycelia discharges spore through soaked with liquid, low temperature induction, room temperature; Microscopy visible zoosporangium and zoospore or the spore that stops, every milliliter of spore content is more than 100,000.
2. liquid-solid composite system cultural method according to claim 1 is characterized in that: the substratum in the described B step is any one in oat, sesame, rye, Semen Phaseoli Vulgaris, lima bean, the soya broth.
3. liquid-solid composite system cultural method according to claim 1 and 2; It is characterized in that: the nutrient solution in the described C step be in the oat, sesame, rye, Semen Phaseoli Vulgaris, lima bean, soybean of 20 ~ 30g any one; After soaking 18 ~ 28 hours in the adding 1000mL zero(ppm) water; Smash tissue to pieces after-filtration; Conventional damp and hot method sterilization, the cigarette root tissue liquid mixing that adds again with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 10 ~ 20g is smashed to pieces in a manner described, filtration sterilization prepares promptly obtains nutrient solution, and said nutrient solution accounts for 30% ~ 50% of vessel volume.
4. liquid-solid composite system cultural method according to claim 1 and 2; It is characterized in that: the nutrient solution in the described C step is any one in 150 ~ 250g fresh carrot, tomato, big bean sprouts, the Semen Phaseoli radiati Germinatus; Add 1000mL and heat up in a steamer water; Smash tissue to pieces after-filtration, more successively through sterilizing behind 0.45 μ m, the 0.22 μ m membrane filtration; Add with the bright root of susceptible cigarette seedling of 6 ~ 7 leaf phases of 10 ~ 20g is smashed to pieces in a manner described, filtration sterilization prepares cigarette root tissue liquid mixing again, said nutrient solution accounts for 30% ~ 50% of vessel volume.
5. liquid-solid composite system cultural method according to claim 1; It is characterized in that: the dull and stereotyped mycelium inoculation of described C step is to cut black shank germ mycelia piece with the scalper of sterilizing in nutrient solution; The mycelia piece size is 2 ~ 4 * 2 ~ 4mm; The solid medium carrier thickness is 1 ~ 2mm, 2 ~ 4 of per 100 milliliters of nutrient solutions inoculation mycelia.
6. liquid-solid composite system cultural method according to claim 1; It is characterized in that: brown shorts that described D step solid medium is particle diameter 1 ~ 3mm or in pea bean straw powder or the silkworm bean straw powder one or more; Compare mixing with brown shorts with the quality of 3 ~ 4:1 ~ 2; The abundant mixing of water of adding 40% ~ 50%, 50% ~ 80% of solid culture fiduciary point vessel volume, conventional moist heat sterilization.
7. according to claim 1 or 6 described liquid-solid composite system cultural methods; It is characterized in that: fresh residue from beans after making that described D step solid medium is particle diameter 1 ~ 3mm and brown shorts compare mixing with the quality of 6 ~ 8:1 ~ 2; Adding water mixture control water cut is 40% ~ 50%; 50% ~ 80% of solid culture fiduciary point vessel volume, conventional moist heat sterilization.
8. liquid-solid composite system cultural method according to claim 1 is characterized in that: described D step liquid mycelium inoculation adds by 3% ~ 5% volume ratio in the solid culture base system, adds the no hydrops in substratum bottom, back.
9. the culture of claim 1 or 8 described liquid-solid composite system cultural methods acquisitions is used for the artificial inoculation thing of black shank, perhaps is used to prepare the additive of band black shank germ sick soil.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396597A (en) * | 2014-12-04 | 2015-03-11 | 云南省烟草农业科学研究院 | Real-time observation method for phytophthora nicotianae infection process |
| CN104396599A (en) * | 2014-12-04 | 2015-03-11 | 云南省烟草农业科学研究院 | Method for phytophthora nicotianae inoculation of tobacco |
| CN104946545A (en) * | 2015-07-14 | 2015-09-30 | 云南省烟草农业科学研究院 | Phytophthora nicotianae culturing method |
| CN110564667A (en) * | 2019-09-25 | 2019-12-13 | 云南省烟草农业科学研究院 | method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1922965A (en) * | 2006-09-05 | 2007-03-07 | 云南省烟草科学研究所 | Sprout period authenticating method for tobacco black shank fastness |
| CN101381685A (en) * | 2008-10-28 | 2009-03-11 | 云南省烟草科学研究所 | Preparation method of tobacco black shank inoculum |
| WO2011110758A1 (en) * | 2010-03-11 | 2011-09-15 | Institut National De La Recherche Agronomique | Treatment of plants against oomycete infection |
-
2012
- 2012-01-31 CN CN2012100216079A patent/CN102559516A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1922965A (en) * | 2006-09-05 | 2007-03-07 | 云南省烟草科学研究所 | Sprout period authenticating method for tobacco black shank fastness |
| CN101381685A (en) * | 2008-10-28 | 2009-03-11 | 云南省烟草科学研究所 | Preparation method of tobacco black shank inoculum |
| WO2011110758A1 (en) * | 2010-03-11 | 2011-09-15 | Institut National De La Recherche Agronomique | Treatment of plants against oomycete infection |
Non-Patent Citations (2)
| Title |
|---|
| 苏振刚等: "烟草黑胫病菌0号生理小种不同条件下培养特性的研究", 《中国烟草科学》 * |
| 马国胜等: "烟草黑胫病菌培养性状的研究", 《中国农业科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396597A (en) * | 2014-12-04 | 2015-03-11 | 云南省烟草农业科学研究院 | Real-time observation method for phytophthora nicotianae infection process |
| CN104396599A (en) * | 2014-12-04 | 2015-03-11 | 云南省烟草农业科学研究院 | Method for phytophthora nicotianae inoculation of tobacco |
| CN104946545A (en) * | 2015-07-14 | 2015-09-30 | 云南省烟草农业科学研究院 | Phytophthora nicotianae culturing method |
| CN110564667A (en) * | 2019-09-25 | 2019-12-13 | 云南省烟草农业科学研究院 | method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores |
| CN110564667B (en) * | 2019-09-25 | 2023-02-28 | 云南省烟草农业科学研究院 | Method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores |
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