CN103571912B - Method for identifying disease resistance of tobacco leaves with brown spot disease - Google Patents
Method for identifying disease resistance of tobacco leaves with brown spot disease Download PDFInfo
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- CN103571912B CN103571912B CN201310540265.6A CN201310540265A CN103571912B CN 103571912 B CN103571912 B CN 103571912B CN 201310540265 A CN201310540265 A CN 201310540265A CN 103571912 B CN103571912 B CN 103571912B
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Abstract
The invention discloses a method for identifying the disease resistance of tobacco leaves with brown spot disease, which belongs to the technical field of tobacco disease resistance identification. The method comprises the following steps: sterilizing and crushing healthy tobacco leaves in a mature period, and preparing a 4% to 6% solution through sterile water; inoculating hypha points of spore production of alternaria alternate to a culture medium flat plate, performing culture in an incubator at the temperature of 24 to 26 DEG C for 10 to 20 days, eluting spores through a tobacco leaf solution after spore production, and performing static culture for 7 to 9 hours to promote germination so as to obtain a bacterium suspension; when the tobacco leaves, which need to be identified, go into the mature period, sampling, and performing sterilization, airing and moisturizing treatment; adding 10 to 16 drips of 10 to 15 micro-milliliter of the bacterium suspension to each tobacco leaf, placing the tobacco leaves into a moisturizing frame or a manual climatic box, and performing illumination culture for 3 to 5 days, wherein the relative humidity of the moisturizing frame or the manual climatic box is equal to or greater than 90 percent, and the temperature of the moisturizing frame or the manual climatic box is 24 to 26 DEG C; after yellow infection spots are formed on the tobacco leaves, measuring the diameter of the spots, and determining the disease resistance. The method is high in success rate, high in reproducibility, quick and accurate in detection, and high in popularization and application value; convenience in checking and monitoring is realized; field pollution caused by artificial inoculation can be avoided.
Description
Technical field
The invention belongs to Tobacco resistance authenticate technology field, it is high to be specifically related to a kind of success ratio, favorable reproducibility, quick and precisely, checks that monitoring is convenient, without the Alternaria alternate blade method of resistance identification of field pollution.
Background technology
Alternaria alternate is a kind of serious plant disease endangering tobacco leaf production, the normal leaf tobacco production in Ge Yan district of China of giving brings huge financial loss, the tobacco bred of the anti-red-star like disease of seed selection is made to seem particularly important, and fast, accurately, reliable and simple and easy to do method of resistance identification effectively can shorten breeding process.In existing authentication method, field Artificial disease nursery method is long for qualification cycle, is subject to grown in field environmental influence, makes effect of inoculation unstable, the quantitative analysis of pathological consequences also exists some problems.Process fast and the diversified feature of applicable object although brown spot pathogen Toxin identification method has, but incomplete same with the actual morbidity course of Alternaria alternate, thus the reliability of qualification result is had an impact, in addition, the determination of actually operating toxin concentration also needs to drop into more manpower and materials.Therefore, develop a kind of success ratio high, favorable reproducibility, quick and precisely, check that monitoring is convenient, without field pollution, and lower-cost Alternaria alternate blade method of resistance identification is a technical problem urgently to be resolved hurrily.
Summary of the invention
The object of the present invention is to provide a kind of success ratio high, favorable reproducibility, quick and precisely, check that detection is convenient, without the Alternaria alternate blade method of resistance identification that field is polluted.
The object of the present invention is achieved like this: a kind of authentication method of Alternaria alternate blade disease resistance, comprises the preparation of tobacco leaf solution, germ Spore cultivation, treats mirror tobacco leaf pre-treatment and Disease Resistance Identification operation, specifically comprise:
Prepared by A, tobacco leaf solution: after ripening stage health is sterilized without the tobacco leaf of scab, dried, carry out historrhexis, then be made into the tobacco leaf solution of 4 ~ 6% with sterilized water;
B, germ Spore cultivation: spore-bearing for brown spot pathogen mycelia point is inoculated on culture medium flat plate, cultivate 10 ~ 20 days for 24 ~ 26 DEG C in incubator, with tobacco leaf solution, spore is washed down after producing spore, continue quiescent culture 7 ~ 9 hours, promote spore germination, obtain brown spot pathogen spore suspension, its spore concentration is 1.5 ~ 2.5 × 10
4individual/ml, percentage of germination>=87%;
C, wait the tobacco leaf pre-treatment of reflecting: sampling when needing the tobacco leaf of the tobacco bred identifying disease resistance to come to the ripening period, after tobacco leaf is sterilized, drying surface solution, then carrying out moisturizing process;
D: Disease Resistance Identification: draw brown spot pathogen spore suspension 10 ~ 15 μ l and drop on tobacco leaf, every sheet tobacco leaf drips 10 ~ 16, then tobacco leaf is placed in relative humidity >=90%, temperature is illumination cultivation 3 ~ 5 days in the moisturizing framework of 24 ~ 26 DEG C or growth cabinet, after tobacco leaf occurs that yellow infects spot, measure lesion diameter, judge the disease resistance of its tobacco leaf of tobacco bred.
The present invention has following beneficial effect:
1, authentication method of the present invention can carry out in indoor, overcome field Artificial disease nursery method due to land for growing field crops natural condition complexity various, the many factors of impact morbidity, is difficult to the shortcoming of accurately manipulation and quantitative analysis, gets final product smooth operation without the need to great many of experiments area, while improving qualification result accuracy, reduce appraisal cost, except having favorable reproducibility, quick and precisely, outside the advantage that conclusion is reliable and stable, also avoid the land for growing field crops pollution because artificial inoculation causes.
2, in brown spot pathogen Spore cultivation step, authentication method of the present invention has taken into account the requirement in sporulation quantity and germination rate two in the selection of substratum.Preferred PDA substratum, Czapek substratum sporulation quantity are large, germination rate is high, coordinate multipoint inoculation and the setting of suitable culture environment, shorten and produce the spore cycle, improve and produce spore synchronism and spore quality stability, while raising determination rates, take into account the reliability of circulation ratio and qualification result.
3, authentication method of the present invention is when preparing brown spot pathogen spore suspension, adds tobacco leaf solution to promote spore germination, and the more common glucose of tobacco leaf solution, sucrose solution contain the abundanter nutrition being conducive to germ spore-germination.Preferably just ripe phase tobacco leaf susceptibility is stronger in institute, can promote infecting of germ spore further, effectively improve the efficiency of qualification, monitoring.
What 4, authentication method of the present invention adopted at sterilisation step is the multiplexing sterilization process of chlorine bleach liquor and ethanolic soln.Described ethanolic soln, while killing tobacco leaf surface microorganism, also has certain extracting effect, is conducive to strengthening brown spot pathogen spore suspension to the perviousness of tobacco leaf, thus has effect that induction is susceptible, improves the success ratio of identification experiment.
5, mirror tobacco leaf pre-treatment step is being treated, the method of the invention selects the tobacco leaf of just ripe phase as qualification sample, object is in order on the basis considering bacteria suspension spore concentration, by accurately controlling the dripping quantity of bacteria suspension, take into account the convenience that the pathogenicity rate of qualification test and scab detect, control the severity of disease development, thus be conducive to the accuracy and the success ratio that improve qualification.
6, during observation after inoculation, monitoring, the method for the invention has all carried out more accurate setting to the temperature of qualification environment, humidity and illumination condition etc.The synergy of each processing parameter especially epidemic disaster, obviously can accelerate sprouting and the infecting blade face thereof of brown spot pathogen spore, further shorten qualification cycle.
In sum, authentication method success ratio of the present invention is high, favorable reproducibility, quick and precisely, checks that monitoring is convenient, can realize without the need to bulk test area, and can not produce the field pollution because artificial inoculation causes, and has good application value.
Embodiment
The present invention is further illustrated below, but limited the present invention never in any form, and based on any conversion that training centre of the present invention is done, the present invention all falls into protection scope of the present invention.
An authentication method for Alternaria alternate blade disease resistance, comprises the preparation of tobacco leaf solution, germ Spore cultivation, treats mirror tobacco leaf pre-treatment and Disease Resistance Identification operation, specifically comprise:
Prepared by A, tobacco leaf solution: after ripening stage health is sterilized without the tobacco leaf of scab, dried, carry out historrhexis, then be made into the tobacco leaf solution of 4 ~ 6% with sterilized water;
B, germ Spore cultivation: spore-bearing for brown spot pathogen mycelia point is inoculated on culture medium flat plate, cultivate 10 ~ 20 days for 24 ~ 26 DEG C in incubator, with tobacco leaf solution, spore is washed down after producing spore, continue quiescent culture 7 ~ 9 hours, promote spore germination, obtain brown spot pathogen spore suspension, its spore concentration is 1.5 ~ 2.5 × 10
4individual/ml, percentage of germination>=87%;
C, wait the tobacco leaf pre-treatment of reflecting: when need the tobacco leaf of the tobacco bred identifying disease resistance enter just the ripe phase time sample, after tobacco leaf is sterilized, dry surface solution, then carry out moisturizing process;
D: Disease Resistance Identification: draw brown spot pathogen spore suspension 10 ~ 15 μ l and drop on tobacco leaf, every sheet tobacco leaf drips 10 ~ 16, then tobacco leaf is placed in relative humidity >=90%, temperature is illumination cultivation 3 ~ 5 days in the moisturizing framework of 24 ~ 26 DEG C or growth cabinet, after tobacco leaf occurs that yellow infects spot, measure lesion diameter, judge the disease resistance of its tobacco leaf of tobacco bred.
The described ripening stage, the healthy tobacco leaf without scab was first ripe phase tobacco leaf.
The tobacco leaf of described just ripe phase is the blade of cigarette strain any leaf.
Described tobacco leaf sterilization refers to the chlorine bleach liquor's immersion 0.8 ~ 1.2min first tobacco leaf being placed in 4 ~ 6%, is placed in the ethanolic soln of 75% again, soaks 20 ~ 40s after clear water rinses.
The chlorine bleach liquor that tobacco leaf is preferably first placed in 5% by the sterilization of described tobacco leaf soaks 1min, is placed in the ethanolic soln of 75% again, soaks 30s after clear water rinses.
What described historrhexis adopted is polishing.
What described inoculation adopted is multiple spot inoculation.
Described multiple spot inoculation refers to each culture medium flat plate inoculation 5 ~ 7 points.
Described substratum can be any one in oat medium, PDA substratum, Czapek substratum.
Described substratum is preferably any one in PDA substratum or Czapek substratum.
Tobacco brown spot pathogen is preferably inoculated on Czapek culture medium flat plate by described germ Spore cultivation, cultivates 15 days for 25 DEG C, is washed down by spore after producing spore with tobacco leaf solution, continue quiescent culture 8 hours, promote germ spore germination in incubator.
Described moisturizing process refers to tobacco leaf to be placed in be had in the apparatus of humidity-holding effect, and the sterilization material which can retain moisture adding nutritive medium is wrapped on petiole.
The described apparatus with humidity-holding effect can be growth cabinet or the framework with humidity-holding effect.
Described nutritive medium can be 10% Huo Shi nutritive medium or 100ppm cigarette compound solution in any one.
Described nutritive medium preferably 10% Huo Shi nutritive medium.
The moisturizing number of days of described moisturizing process is 3 ~ 5 days.
Describedly drop in front tobacco leaf referring to and drops in blade, not on master pulse.
After described absorption brown spot pathogen spore suspension drops on tobacco leaf, tobacco leaf being placed in relative humidity is 95%, and temperature is illumination cultivation 4 days in the moisturizing framework of 25 DEG C or growth cabinet.
Described measurement lesion diameter, judge that the disease resistance of its tobacco leaf of tobacco bred refers to each test and all must add clean leaf Huang as disease-resistant contrast, G140 is as susceptible contrast, variance analysis is carried out after the tobacco leaf lesion diameter of the tobacco bred participating in qualification is measured, be disease-resistant grade with its blade disease resistance of the inapparent tobacco bred of the yellow difference of clean leaf, be Infection Grades with its blade disease resistance of the inapparent tobacco bred of G140 difference, its blade disease resistance of tobacco bred between clean leaf between yellow and G140 be in anti-or middle sense grade.
embodiment 1
---NC89 kind is to the Disease Resistance Identification of Alternaria alternate
Prepared by A, tobacco leaf solution: after being plucked by healthy for the first ripe phase tobacco leaf without scab, the chlorine bleach liquor being first placed in 5% soaks 1min, is placed in the ethanolic soln of 75% after clear water rinses again, soaks 30s.Levigate in the mortar of sterilizing after drying, carry out historrhexis, be made into the tobacco leaf solution of 5% with sterilized water;
B, germ Spore cultivation: by culture dish length have the mycelia of brown spot pathogen sterilization blade to be cut into small pieces, then be transferred on Czapek culture medium flat plate, each plating 6 points.The incubator being placed in 25 DEG C is cultivated 15 days, is washed down by spore after producing spore with tobacco leaf solution, continues quiescent culture 8 hours, and promote spore germination, obtain brown spot pathogen spore suspension, its spore concentration is 2.3 × 10
4individual/ml, percentage of germination is 88%;
C, until mirror tobacco leaf pre-treatment: after the tobacco leaf of the tobacco bred needing qualification comes to the ripening period, sample when middle part blade is just ripe.First chlorine bleach liquor tobacco leaf being placed in 5% soaks 1min, is placed in the ethanolic soln of 75% after clear water rinses again, soaks 30s, dries surface solution, and be wrapped on petiole by the sterilization cotton of the Huo Shi nutritive medium adding 10%.
D, Disease Resistance Identification: draw brown spot pathogen spore suspension 12 μ l with liquid-transfering gun and drop in position between the vein of tobacco leaf front, every sheet tobacco leaf drips 14, then tobacco leaf being placed in relative humidity is 95%, temperature is cultivate 4 days in the moisturizing framework of 25 DEG C, after tobacco leaf occurs that yellow infects spot, measure lesion diameter, judge the disease resistance of its tobacco leaf of tobacco bred.The clean leaf Huang of Disease Resistance Identification does disease-resistant contrast, does susceptible contrast with G140.
Qualification result shows: the lesion diameter of NC89 kind tobacco leaf surface is 1.7 ~ 2.0cm, shows as Infection Grades to Alternaria alternate.
embodiment 2
---PVH01 and PVH06 kind is to the Disease Resistance Identification of Alternaria alternate
Prepared by A, tobacco leaf solution: after being plucked by healthy for the first ripe phase tobacco leaf without scab, the chlorine bleach liquor being first placed in 6% soaks 0.8min, is placed in the ethanolic soln of 75% after clear water rinses again, soaks 20s.Levigate in the mortar of sterilizing after drying, carry out historrhexis, be made into the tobacco leaf solution of 4% with sterilized water;
B, germ Spore cultivation: by culture dish length have the mycelia of brown spot pathogen sterilization blade to be cut into small pieces, then be transferred on Czapek culture medium flat plate, each plating 7 points.The incubator being placed in 24 DEG C is cultivated 10 days, is washed down by spore after producing spore with tobacco leaf solution, continues quiescent culture 7 hours, and promote spore germination, obtain brown spot pathogen spore suspension, its spore concentration is 1.8 × 10
4individual/ml, percentage of germination is 87%;
C, until mirror tobacco leaf pre-treatment: after the tobacco leaf of the tobacco bred needing qualification comes to the ripening period, sample when upper two canopy portion blades are just ripe.First chlorine bleach liquor tobacco leaf being placed in 6% soaks 0.8min, is placed in the ethanolic soln of 75% after clear water rinses again, soaks 20s, dries surface solution, and be wrapped on petiole by the sterilization cotton of the cigarette compound solution adding 100ppm.
D, Disease Resistance Identification: draw brown spot pathogen spore suspension 15 μ l with liquid-transfering gun and drop in position between the vein of tobacco leaf front, every sheet tobacco leaf drips 10, then tobacco leaf being placed in relative humidity is 90%, temperature is cultivate 3 days in the moisturizing framework of 26 DEG C, after tobacco leaf occurs that yellow infects spot, measure lesion diameter, judge the disease resistance of its tobacco leaf of tobacco bred.The clean leaf Huang of Disease Resistance Identification does disease-resistant contrast, does susceptible contrast with G140.
Qualification result shows: the lesion diameter of PVH01, PVH06 kind tobacco leaf surface is respectively 1.9 ~ 2.1cm and 1.8 ~ 2.1cm, and two kinds show as Infection Grades to Alternaria alternate.
embodiment 3
---9147,9102,9111-21 new lines is to the Disease Resistance Identification of Alternaria alternate
Prepared by A, tobacco leaf solution: after being plucked by healthy for the first ripe phase tobacco leaf without scab, the chlorine bleach liquor being first placed in 4% soaks 1.2min, is placed in the ethanolic soln of 75% after clear water rinses again, soaks 40s.Levigate in the mortar of sterilizing after drying, carry out historrhexis, be made into the tobacco leaf solution of 6% with sterilized water;
B, germ Spore cultivation: by culture dish length have the mycelia of brown spot pathogen sterilization blade to be cut into small pieces, then be transferred on PDA culture medium flat plate, each plating 5 points.The incubator being placed in 26 DEG C is cultivated 20 days, is washed down by spore after producing spore with tobacco leaf solution, continues quiescent culture 9 hours, and promote spore germination, obtain brown spot pathogen spore suspension, its spore concentration is 2.1 × 10
4individual/ml, percentage of germination is 90%;
C, until mirror tobacco leaf pre-treatment: after the tobacco leaf of the tobacco bred needing qualification comes to the ripening period, sample when upper two canopy portion blades are just ripe.First chlorine bleach liquor tobacco leaf being placed in 4% soaks 1.2min, is placed in the ethanolic soln of 75% after clear water rinses again, soaks 40s, dries surface solution, and be wrapped on petiole by the sterilization cotton of the cigarette compound solution adding 100ppm.
D, Disease Resistance Identification: draw brown spot pathogen spore suspension 10 μ l with liquid-transfering gun and drop in position between the vein of tobacco leaf front, every sheet tobacco leaf drips 16, then tobacco leaf being placed in relative humidity is 92%, temperature is cultivate 5 days in the moisturizing framework of 24 DEG C, after tobacco leaf occurs that yellow infects spot, measure lesion diameter, judge the disease resistance of its tobacco leaf of tobacco bred.The clean leaf Huang of Disease Resistance Identification does disease-resistant contrast, does susceptible contrast with G140.
9147,9102 qualification result shows:, the lesion diameter of 9111-21 tri-new lines tobacco leaf surfaces is respectively 1.4 ~ 1.8cm, 1.4 ~ 1.7cm, 1.0 ~ 1.3cm, three new lines show as 9147 and 9102 to Alternaria alternate and show as Infection Grades, and 9111-21 shows as disease-resistant grade.
Claims (5)
1. an authentication method for Alternaria alternate blade disease resistance, is characterized in that comprising the preparation of tobacco leaf solution, germ Spore cultivation, treats mirror tobacco leaf pre-treatment and Disease Resistance Identification operation, specifically comprise:
Prepared by A, tobacco leaf solution: after healthy for the ripening stage first ripe phase tobacco leaf sterilization without scab, drying, carry out historrhexis, then be made into the tobacco leaf solution of 4 ~ 6% with sterilized water; Described sterilization be first tobacco leaf is placed in 4 ~ 6% chlorine bleach liquor soak 0.8 ~ 1.2min, be placed in the ethanolic soln of 75% after clear water rinses again, soak 20 ~ 40s;
B, germ Spore cultivation: spore-bearing for brown spot pathogen mycelia point is inoculated on PDA substratum or Czapek culture medium flat plate, cultivate 10 ~ 20 days for 24 ~ 26 DEG C in incubator, with tobacco leaf solution, spore is washed down after producing spore, continue quiescent culture 7 ~ 9 hours, promote spore germination, obtain brown spot pathogen spore suspension, its spore concentration is 1.5 ~ 2.5 × 10
4individual/ml, percentage of germination>=87%;
C, wait the tobacco leaf pre-treatment of reflecting: sampling when needing the tobacco leaf of the tobacco bred identifying disease resistance to come to the ripening period, after tobacco leaf is sterilized, drying surface solution, then carrying out moisturizing process; Described sterilization be first tobacco leaf is placed in 4 ~ 6% chlorine bleach liquor soak 0.8 ~ 1.2min, be placed in the ethanolic soln of 75% after clear water rinses again, soak 20 ~ 40s;
D: Disease Resistance Identification: draw brown spot pathogen spore suspension 10 ~ 15 μ l and drop on tobacco leaf, every sheet tobacco leaf drips 10 ~ 16, then tobacco leaf is placed in relative humidity >=90%, temperature is illumination cultivation 3 ~ 5 days in the moisturizing framework of 24 ~ 26 DEG C or growth cabinet, after tobacco leaf occurs that yellow infects spot, measure lesion diameter, judge the disease resistance of its tobacco leaf of tobacco bred.
2. the authentication method of Alternaria alternate blade disease resistance as claimed in claim 1, is characterized in that moisturizing process described in step C refers to tobacco leaf to be placed in and has in the apparatus of humidity-holding effect, and the sterilization material which can retain moisture adding nutritive medium is wrapped on petiole.
3. the authentication method of Alternaria alternate blade disease resistance as claimed in claim 2, is characterized in that described nutritive medium is the Huo Shi nutritive medium of 10% or the cigarette compound solution of 100ppm.
4. the authentication method of Alternaria alternate blade disease resistance as claimed in claim 1, after it is characterized in that drawing brown spot pathogen spore suspension described in D step drops on tobacco leaf, tobacco leaf being placed in relative humidity is 95%, and temperature is illumination cultivation 4 days in the moisturizing framework of 25 DEG C or growth cabinet.
5. the authentication method of Alternaria alternate blade disease resistance as claimed in claim 1, it is characterized in that measuring lesion diameter described in D step, judge that the disease resistance of its tobacco leaf of tobacco bred refers to each test and all must add clean leaf Huang as disease-resistant contrast, G140 is as susceptible contrast, variance analysis is carried out after the tobacco leaf lesion diameter of the tobacco bred participating in qualification is measured, be disease-resistant grade with its blade disease resistance of the inapparent tobacco bred of the yellow difference of clean leaf, be Infection Grades with its blade disease resistance of the inapparent tobacco bred of G140 difference, its blade disease resistance of tobacco bred between clean leaf Huang and G140 resists or middle sense grade in being.
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| CN104082055A (en) * | 2014-06-17 | 2014-10-08 | 贵州省烟草科学研究院 | Method for high-flux identification of ralstonia solanacearum resistance |
| CN105567784A (en) * | 2016-02-05 | 2016-05-11 | 云南省烟草农业科学研究院 | Method for rapidly and accurately identifying brown spot resistance of tobacco variety |
| CN105660222B (en) * | 2016-03-23 | 2019-04-16 | 河南农业大学 | A kind of inoculation identification method that can observe black embryo of wheat disease symptoms in time |
| CN112080434B (en) * | 2020-08-28 | 2022-05-10 | 中国农业科学院烟草研究所 | Artificial inoculation identification method for alternaria alternata |
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| CN101519645A (en) * | 2009-04-01 | 2009-09-02 | 云南省烟草科学研究所 | Preparation method of alternaria alternate spore inoculum |
| CN103194421A (en) * | 2013-03-22 | 2013-07-10 | 云南省烟草农业科学研究院 | Alternaria alternate spore suspension liquid and application thereof |
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| CN101519645A (en) * | 2009-04-01 | 2009-09-02 | 云南省烟草科学研究所 | Preparation method of alternaria alternate spore inoculum |
| CN103194421A (en) * | 2013-03-22 | 2013-07-10 | 云南省烟草农业科学研究院 | Alternaria alternate spore suspension liquid and application thereof |
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