CN110169327B - Method for establishing peanut bacterial wilt resistance identification garden - Google Patents
Method for establishing peanut bacterial wilt resistance identification garden Download PDFInfo
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- CN110169327B CN110169327B CN201910497711.7A CN201910497711A CN110169327B CN 110169327 B CN110169327 B CN 110169327B CN 201910497711 A CN201910497711 A CN 201910497711A CN 110169327 B CN110169327 B CN 110169327B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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Abstract
The invention discloses a method for establishing a peanut bacterial wilt resistance identification nursery, which comprises the steps of separating and extracting and culturing a ralstonia solanacearum solution from peanut bacterial wilt strains, wherein the concentration of the ralstonia solanacearum solution is 3-5 multiplied by 107Soaking diseased peanut seeds in cfu/ml ralstonia solanacearum liquid, wherein the soaking amount is 10 peanut seeds/5 ml, and planting after soaking for 30 min; spraying the ralstonia solanacearum liquid on the leaf surfaces of the peanuts after the peanuts grow seedlings, spraying the ralstonia solanacearum liquid once every 10 days, damaging the leaves, and inoculating ralstonia solanacearum again; pulling out the dead peanut plants caused by the bacterial wilt, crushing and spraying the crushed plants in the field, re-planting infected peanut seeds infected with the bacterial wilt in the peanut inter-row space and dead seedling empty space, continuing injuring leaves and inoculating, dead seedling and reseeding until harvesting after seedling emergence, crushing the diseased plants and returning the crushed plants to the field; and (4) planting in spring and autumn every year for two seasons, replanting dead seedlings for 2-4 times, and circulating the steps for 2-3 years to form the peanut bacterial wilt resistance identification nursery. Solves the problems of long establishing period, time and labor waste and the like of the peanut field disease-resistant identification nursery.
Description
Technical Field
The invention belongs to the field of bacterial wilt resistance identification, and relates to a method for establishing a peanut bacterial wilt resistance identification nursery.
Background
Bacterial wilt is one of important diseases in peanut producing areas in south China, the field morbidity is generally 10-30%, and in severe cases, the field morbidity reaches more than 50%. Besides causing yield reduction, the method also has important influence on the nutritional quality of seeds, aflatoxin pollution and the like. The breeding of the bacterial wilt-resistant variety is the most effective method for improving the yield of the peanuts, disease-resistant resources are required for disease-resistant breeding as breeding materials, the existing resource resistance identification and screening technology mainly depends on identification of a field disease nursery, and the establishment period is long and time and labor are wasted due to the reasons of uneven disease occurrence, low disease incidence and the like of the field natural disease nursery. Therefore, it is very necessary to find a fast, effective and stable method for establishing a bacterial wilt nursery.
Disclosure of Invention
The invention aims to provide a method for establishing a peanut bacterial wilt resistance identification garden, which aims to solve the problems of long establishment period, time waste and labor waste of a peanut field disease resistance identification garden.
The technical scheme adopted by the invention is that the method for establishing the peanut bacterial wilt resistance identification nursery comprises the following specific steps:
s1, separating, extracting and culturing ralstonia solanacearum liquid;
step S2, carrying out green dead bacteria liquid immersion planting: soaking the infected peanut seeds with the ralstonia solanacearum liquid, and planting after seed soaking;
step S3, wounded leaf inoculation: spraying the ralstonia solanacearum liquid on the leaf surfaces of the peanuts after the peanuts grow seedlings, damaging the leaves and inoculating ralstonia solanacearum again;
step S4, filling seeds with dead seedlings: pulling out dead peanut seedlings which are withered and withered due to bacterial wilt, crushing and spraying the crushed peanut seedlings in the field, re-planting infected peanut seeds infected with ralstonia solanacearum through seed soaking in peanut inter-row and dead seedling empty spaces, continuing injuring leaves and inoculating, killing seedlings and supplementing seeds after seedling emergence until harvesting, crushing diseased plants and returning the crushed plants to the field;
and S5, planting the peanuts in spring and autumn for two seasons each year, and circulating the steps for 2-3 years to form the peanut bacterial wilt resistance identification garden.
Further, the adding amount of the ralstonia solanacearum liquid in the step S2 is 10 peanut seeds/5 ml.
Further, the concentration of the ralstonia solanacearum liquid in the steps S2 and S4 is 3-5 × 107 cfu/ml; the seed soaking time of the step S2 is 30 min.
Further, the step S3 of inoculating the wounded leaves is performed every 10 days; and in the step S4, the dead seedlings are replanted for 2-4 times every year.
Further, the separation and extraction of the ralstonia solanacearum liquid in the step S1 is realized by the following steps: the main root of a seedling peanut plant which appears 40-60 days old and begins to wilt from a peanut bacterial wilt nursery is rat-tail-shaped, the root tip is soaked in water, lateral roots are not developed, the root of the wilted peanut plant is soaked in alcohol in an aseptic environment, then flame burning and sterilizing are carried out, the main root is cut off from 1/3, the plant is placed and soaked in sterilized water with a downward cut for 3-5 min, and the phenomenon that white fungus pus overflows from the cut is observed to obtain a ralstonia liquid for culture.
Further, the step S1 of culturing the ralstonia solanacearum solution specifically includes the following steps:
step S11, preparing a solid culture medium and a liquid culture medium: peeling and slicing potatoes, weighing 60g and 10g of potatoes, 60g and 10g of cane sugar and 7.5g of agar, dicing, boiling and softening 10g of potatoes, adding 10g of cane sugar and 7.5g of agar, adding water to a constant volume of 500ml, and filtering to obtain a solid culture medium; dicing 60g of potato, putting into 3L of water, boiling to soften, adding 60g of cane sugar, fixing the volume to 3L, filtering to obtain a liquid culture medium, sterilizing the solid culture medium and the liquid culture medium at 121 ℃ for 25min, and storing in a refrigerator;
step S12, preparing a TTC solution; weighing 0.1g of 2,3, 5-triphenyltetrazolium chloride, dissolving with purified water, diluting to a constant volume of 10ml to obtain TTC solution, and shading and refrigerating;
step S13, taking out 2 solid culture mediums and 9 liquid culture mediums, sterilizing for two hours, putting the solid culture mediums into an oven, preserving heat, disassembling culture dishes, pouring the solid culture mediums into 6 culture dishes in sequence, wherein the height of the solid culture mediums is half of the height in the culture dishes, adding 6.5ml of TTC solution into the rest solid culture mediums, shaking uniformly, pouring the TTC solution into the 6 culture dishes in sequence, after solidification, staining the ralstonia solanacearum liquid extracted and separated previously with toothpicks, lightly scribing on the culture dishes, sealing the culture dishes with sealing adhesive tapes, and putting the culture dishes in the 28 ℃ culture box for 2 days; when pink single bacterial colony appears in the culture dish, the liquid culture medium is poured into a centrifuge tube, the bacterial colony in the culture dish is picked by a toothpick and then is put into the centrifuge tube, and the centrifuge tube is put into a shaking table with the rotating speed of 150r/min and the temperature of 28 ℃ for the culture of the ralstonia solanacearum liquid.
Further, the step S5 is to form a disease-resistant identification nursery with the disease incidence rate of susceptible varieties being more than 90%.
The method has the beneficial effect of quickly establishing the peanut bacterial wilt-resistant identification garden. The conventional peanut field disease-resistant identification garden is established by accumulating pathogenic bacteria in a continuous cropping plot through natural continuous cropping peanuts so as to form a disease-resistant identification garden with serious disease, and the establishment period needs 5-6 years; according to the method, the bacterial wilt pathogenic bacteria are continuously and circularly inoculated in modes of seed soaking, leaf damage, reseeding, disease returning and the like, so that the bacterial wilt pathogenic bacteria in the continuous cropping peanut field are multiplied and accumulated, the establishment period of the disease-resistant identification garden for the bacterial wilt of the peanut can be obviously shortened, the disease-resistant identification garden can be established only in 2-3 years, the disease-resistant identification garden with the disease incidence rate of more than 90% of a disease-susceptible variety is formed, and the problems of long establishment period, time waste and labor waste of the disease-resistant identification garden in the peanut field are solved. Has important significance for breeding disease-resistant peanut varieties and identifying disease resistance of progeny materials.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The method for establishing the peanut bacterial wilt resistance identification garden comprises the following specific steps:
and step S1, identifying the pathogenicity of the ralstonia solanacearum and culturing the ralstonia solanacearum to obtain ralstonia solanacearum liquid.
And (3) separating and extracting ralstonia solanacearum: placing purified water into a sterilizing pot, sterilizing for 2 hours, taking out (sterilized water), and preparing for extracting the ralstonia solanacearum liquid. Taking the peanut withered plants which begin to wither from the peanut plantation, wherein the main roots are rat tail-shaped, the root tips are soaked in water, and the lateral roots are not developed. The picked withered peanut plants are cut off branches, leaves and whiskers. Sterile water, a centrifuge tube, scissors, a marker pen, tweezers, cotton and 75% alcohol are prepared, and the superclean bench is taken out for ultraviolet disinfection for 30 minutes. The material was placed in a clean bench, soaked in alcohol with cotton, and the hands were wiped. Slightly soaking the roots of the diseased plants in alcohol in an ultra-clean workbench, burning and sterilizing by flame, cutting off 1/3 parts of main roots away from the root ends, soaking the plants in a test tube (1-2 plants/2 ml liquid in 40-60 days of seedling age) with a downward cut in sterile water, standing for 3-5 min, and observing the white pyogenic bacteria overflowing from the cut.
Preparing solid and liquid culture media: peeling and slicing potatoes, and weighing 60g and 10g of potatoes, 60g and 10g of cane sugar and 7.5g of agar. Boiling 10g of diced potato, softening, adding 10g of sucrose and 7.5g of agar, diluting to 500ml with water, filtering, and placing into a conical flask to obtain the solid culture medium. Putting 60g of diced potato into a pot filled with 3L of water, boiling for softening, adding 60g of cane sugar, fixing the volume to 3L, filtering into a conical flask to obtain a liquid culture medium, sterilizing the liquid culture medium in a sterilization pot at 121 ℃ for 25min, and then putting the liquid culture medium in a refrigerator for cold storage.
Preparing a TTC solution: preparing 10ml of TTC solution (color developing agent), weighing 0.1g of TTC (2, 3, 5-triphenyltetrazolium chloride), dissolving with a small amount of purified water, fixing the volume to 10ml with a volumetric flask, putting into a centrifuge tube (convenient for shading and preserving), taking purified water by using a liquid transfer gun, pouring into the centrifuge tube, wrapping the TTC solution by using tinfoil paper, putting into a refrigerator, shading, and filtering and preserving in an ultraclean workbench. The pathogens could show a red color in TTC to distinguish counts.
Culturing a ralstonia solanacearum solution: taking out 2 solid culture media and 9 liquid culture media from the refrigerator, placing the solid culture media into a sterilization pot, sterilizing for two hours, taking out the solid culture media, and placing the solid culture media into an oven for heat preservation. Placing the alcohol burner, the culture dish, the glass long tube, the toothpick, the liquid-transferring gun and the gun head into a superclean bench for ultraviolet sterilization for 30 min. After the superclean workbench is disinfected, the solid culture medium is taken and placed into the superclean workbench, after both hands are disinfected by alcohol, the alcohol lamp is ignited, and the culture dish is disassembled in the superclean workbench. And sequentially pouring the solid culture medium into 6 culture dishes, wherein the height of the culture medium is half of the height in the dishes. Adding 6.5ml of TTC solution into the residual culture medium in the solid culture medium conical flask, shaking uniformly, then pouring into 6 culture dishes in sequence, after solidification, dipping the ralstonia solanacearum liquid extracted and separated previously with a toothpick, and lightly scribing on the culture dishes without using large force to prevent puncturing the culture medium. Sealing the culture dish with sealing adhesive tape after marking, the adhesive tape is to be butt-jointed end to prevent the entry of miscellaneous bacteria, and placing the culture dish upside down to prevent vapor from running upwards, picking up the desktop, and closing the super clean bench. Put into 28 ℃ incubator in 2 days or so and appear pink single bacterial colony in the culture dish, pour liquid medium into the centrifuging tube, about 2/3's volume, centrifuging tube and culture dish are put on one side, place in the another side after the alcohol burner lights, press from both sides with tweezers and get a toothpick, after disinfecting on the alcohol burner, open the culture dish, put into the centrifuging tube after picking bacterial colony in the culture dish with the toothpick, put into the shaking table with the centrifuge tube after accomplishing required volume, the shaking table rotational speed is 150r/min, the temperature is 28 ℃, notice must be sticky and prevent dropping. After about one day, if the toothpicks in the centrifugal tube are hidden and appeared, the bacteria liquid concentration meets the requirement. The liquid culture medium has the function of enabling bacterial colonies in solid culture to grow and propagate in liquid, and aims to increase the number of the bacterial colonies, enable the liquid to be convenient for soaking seeds and infecting peanut seeds, enable the bacterial liquid to be clearly visible when toothpicks are just put in the bacterial liquid, enable the bacteria to increase along with the extension of culture time, enable the bacterial liquid to be turbid, enable the toothpicks to be hidden and appear, and enable the toothpicks to be unclear when the bacteria increase.
Step S2, carrying out green dead bacteria liquid immersion planting: pouring the ralstonia solanacearum liquid into prepared 36 peanut seeds of sweet osmanthus flowers for soaking for 30min according to the addition amount of about 10 peanuts/5 ml, and taking out the peanuts for planting after the seed soaking is finished. Can not be exposed in the sun, and the bacterial wilt germs can be killed by the exposure.
The concentration of the ralstonia solanacearum liquid is about 3 multiplied by 10 of ralstonia solanacearum in each milliliter of the bacterial liquid7~5×107And (4) respectively.
Using 3X 107~5×107 The peanut seeds are soaked in the bacterial wilt liquid of cfu/ml for 30min, the incidence rate of bacterial wilt is highest, the incidence time is shortest, and the rapid establishment of a disease-resistant identification nursery is facilitated.
And step S3, spraying the ralstonia solanacearum liquid on the peanut leaf surfaces after the peanuts emerge, damaging the leaves, inoculating ralstonia solanacearum again, and inoculating once every 10 days.
The seed soaking before planting is from the infection of seed part, the damage to the leaf is the infection of leaf, and the spraying of ralstonia liquid is used for inoculation after seedling emergence, so as to carry out double infection on the peanut, aggravate the incidence of disease of the peanut and further increase the pathogenic bacteria of the peanut planting field.
And step S4, after the peanuts are infected with pathogenic bacteria, the wilt and death of the seedlings are caused, the withered and death seedlings are pulled out, the withered and death seedlings are crushed and sprayed in the field, and seeds infected with the ralstonia solanacearum are planted again among the peanut lines and in the dead seedling empty space, the damaged leaves are inoculated, and the dead seedlings are replanted.
The method is characterized in that the seeds infected with germs are planted again in the dead seedling open space after the seedlings are killed, the method is equivalent to reseeding, the withered and dead seedlings are pulled out, crushed and sprayed in the field, the more pathogens are infected in the field, the pathogens are infected in a circulating mode, and the pathogenic effect on peanuts is better.
And S5, circulating the steps for 2-3 years, planting peanuts in two seasons in spring and autumn every year, and replanting dead seedlings for 2-4 times to finally form the peanut bacterial wilt resistance identification nursery.
The test result is compared with the occurrence situation of peanut bacterial wilt in a normal peanut continuous cropping field (the comparison does not include the steps of seed soaking, pathogen inoculation, leaf injury and seedling death after cyclic planting, only the conventional seed is harvested, the second-season planting is different from the conventional method, the conventional method is to harvest and then plant the peanut seedlings after 4-5 months, the method of the invention plants the peanut seedlings about 2 months after death, namely, the peanut seedlings can be replanted for 2-4 times in spring or autumn, and the peanut seedlings withered once in 1-2 months, namely, the peanut seedlings are replanted once in 1-2 months.
The conventional field disease-resistant identification nursery is a nursery which is planted conventionally, does not soak seeds, inoculates pathogenic bacteria, damages leaves and dies, and is planted in a circulating mode after being mature and harvested for 5 months, and a disease nursery with serious disease can be formed in 5-6 years in the south. The method comprises the steps of soaking seeds before planting, spraying the ralstonia solanacearum liquid on the leaf surfaces of peanuts after emergence of seedlings, damaging the leaves, inoculating ralstonia solanacearum again, inoculating once every 10 days, pulling out dead seedlings along with roots after dead seedlings, replanting, planting once again after dead seedlings for about 2 months, crushing the dead seedlings, spraying the crushed seedlings in the field, and harvesting. Two-season peanuts are planted in spring and autumn every year, a disease-resistant identification garden with disease-susceptible varieties with the disease rate of more than 90% is formed in a circulating mode for 2-3 years, as shown in table 1, the disease rate of peanut bacterial wilt only reaches 51.3% after the disease nursery of natural continuous cropping peanuts are continuously cropped for 3 years, the disease rate of peanut bacterial wilt can reach 94.2% after the disease nursery of continuous cropping is inoculated with germs and reseeded, and the identification effect is obvious.
TABLE 1 occurrence of peanut bacterial wilt during establishment of peanut bacterial wilt nursery
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (5)
1. The method for establishing the peanut bacterial wilt resistance identification garden is characterized by comprising the following specific steps of:
s1, finding peanut plants withered due to peanut bacterial wilt from peanut fields of 40-60 days, enabling main roots to be rat-tail-shaped, enabling root tips to be water-soaked and lateral roots to be undeveloped, soaking roots of the wilted peanut plants in alcohol in an aseptic environment, burning and sterilizing the soaked peanut plants by flame, cutting off the peanut plants at positions 1/3 away from root ends of the main roots, standing and soaking the plants in sterilized water for 3-5 min with downward cuts, and observing that white bacterial pus overflows from the cuts to obtain ralstonia liquid and culturing the ralstonia liquid;
the method comprises the following specific steps of:
step S11, preparing a solid culture medium and a liquid culture medium: peeling and slicing potatoes, weighing 60g and 10g of potatoes, 60g and 10g of cane sugar and 7.5g of agar, dicing, boiling and softening 10g of potatoes, adding 10g of cane sugar and 7.5g of agar, adding water to a constant volume of 500mL, and filtering to obtain a solid culture medium; dicing 60g of potato, putting into 3L of water, boiling to soften, adding 60g of cane sugar, fixing the volume to 3L, filtering to obtain a liquid culture medium, sterilizing the solid culture medium and the liquid culture medium at 121 ℃ for 25min, and storing in a refrigerator;
step S12, preparing a TTC solution; weighing 0.1g of 2,3, 5-triphenyltetrazolium chloride, dissolving with purified water, diluting to a constant volume of 10mL to obtain TTC solution, and shading and refrigerating;
step S13, taking out 2 solid culture mediums and 9 liquid culture mediums, sterilizing for two hours, putting the solid culture mediums into an oven, preserving heat, disassembling culture dishes, pouring the solid culture mediums into 6 culture dishes in sequence, wherein the height of the solid culture mediums is half of the height in the culture dishes, adding 6.5mL of TTC solution into the rest solid culture mediums, shaking uniformly, pouring the TTC solution into the 6 culture dishes in sequence, after solidification, staining the ralstonia solanacearum liquid extracted and separated previously with toothpicks, lightly scribing on the culture dishes, sealing the culture dishes with sealing adhesive tapes, and putting the culture dishes in the 28 ℃ culture box for 2 days; when pink single bacterial colony appears in the culture dish, pouring the liquid culture medium into a centrifuge tube, picking the bacterial colony in the culture dish by using a toothpick, putting the bacterial colony into the centrifuge tube, putting the centrifuge tube into a shaking table with the rotating speed of 150r/min and the temperature of 28 ℃, and carrying out ralstonia liquid culture;
step S2, carrying out green dead bacteria liquid immersion planting: soaking the infected peanut seeds with the ralstonia solanacearum liquid, and planting after seed soaking;
step S3, wounded leaf inoculation: spraying the ralstonia solanacearum liquid on the leaf surfaces of the peanuts after the peanuts grow seedlings, damaging the leaves and inoculating ralstonia solanacearum again;
step S4, filling seeds with dead seedlings: pulling out dead peanut seedlings which are withered and withered due to bacterial wilt, crushing and spraying the crushed dead peanut seedlings in a field, re-planting infected peanut seeds infected with ralstonia solanacearum in the peanut inter-row space and dead peanut empty space, continuing injuring leaves and inoculating after seedling emergence, and replanting dead seedlings until harvesting, crushing diseased plants and returning to the field;
and S5, planting the peanuts in spring and autumn for two seasons each year, and circulating the steps for 2-3 years to form the peanut bacterial wilt resistance identification garden.
2. The method for establishing the peanut bacterial wilt resistance identification nursery as claimed in claim 1, wherein the amount of the ralstonia solanacearum added in step S2 is 10 peanut seeds/5 mL.
3. The method for establishing the peanut bacterial wilt resistance identification nursery as claimed in claim 1, wherein the concentration of the ralstonia solanacearum liquid in steps S2 and S4 is 3-5 x 107cfu/mL, and the seed soaking time of the step S2 is 30 min.
4. The method for establishing the peanut bacterial wilt resistance identification nursery as claimed in any one of claims 1 to 3, wherein the step S3 of leaf wounding inoculation is performed once every 10 days, and the step S4 of seedling death and reseeding is performed 2 to 4 times per year.
5. The method for establishing the peanut bacterial wilt resistance identification nursery as claimed in any one of claims 1 to 3, wherein step S5 is implemented to form a disease resistance identification nursery with disease incidence rate of susceptible varieties greater than 90%.
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| CN114651689A (en) * | 2022-03-17 | 2022-06-24 | 福建农林大学 | Peanut bacterial wilt field initial flowering stage inoculation identification method |
| CN114747445A (en) * | 2022-04-29 | 2022-07-15 | 湖北恩施中国南方马铃薯研究中心 | Method for establishing potato bacterial wilt resistance identification garden |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102835288A (en) * | 2012-08-28 | 2012-12-26 | 新疆农垦科学院 | Method for establishing disease garden to identify soil-borne diseases |
| KR20130054545A (en) * | 2011-11-17 | 2013-05-27 | 대한민국(농촌진흥청장) | Steip gene enhancing resistance to bacterial wilt and use thereof |
| CN105359855A (en) * | 2015-10-19 | 2016-03-02 | 中国农业科学院棉花研究所 | Improved verticillium wilt resistance identification method for cotton variety in artificial disease garden during whole growth period |
| CN107211861A (en) * | 2017-07-04 | 2017-09-29 | 四川省农业科学院经济作物育种栽培研究所 | The method of seedling stage Rapid identification tobacco bred Resistance to bacterial wilt |
| CN108243891A (en) * | 2018-04-17 | 2018-07-06 | 广西壮族自治区农业科学院经济作物研究所 | A kind of disease-resistant peanut cultivation method |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6963020B1 (en) * | 1999-04-30 | 2005-11-08 | National Institute Of Agrobiological Sciences | DNA polymorphism-based methods for identifying field resistance of rice to rice blast |
| CN101496481B (en) * | 2009-03-09 | 2011-06-15 | 云南省烟草科学研究所 | Method for identifying resistance to tobacco bacterial wilt during seedling stage |
| US11261458B2 (en) * | 2017-07-28 | 2022-03-01 | Two Blades Foundation | Potyvirus resistance genes and methods of use |
| CN108739130A (en) * | 2018-05-17 | 2018-11-06 | 广东省农业科学院植物保护研究所 | A kind of method of greenhouse seedling stage assay pumpkin Resistance to bacterial wilt kind |
| CN109055483A (en) * | 2018-08-28 | 2018-12-21 | 四川省农业科学院经济作物育种栽培研究所 | A kind of crop field inoculation Resistance Identification method of peanut sclerotium rolfsii |
-
2019
- 2019-06-10 CN CN201910497711.7A patent/CN110169327B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130054545A (en) * | 2011-11-17 | 2013-05-27 | 대한민국(농촌진흥청장) | Steip gene enhancing resistance to bacterial wilt and use thereof |
| CN102835288A (en) * | 2012-08-28 | 2012-12-26 | 新疆农垦科学院 | Method for establishing disease garden to identify soil-borne diseases |
| CN105359855A (en) * | 2015-10-19 | 2016-03-02 | 中国农业科学院棉花研究所 | Improved verticillium wilt resistance identification method for cotton variety in artificial disease garden during whole growth period |
| CN107211861A (en) * | 2017-07-04 | 2017-09-29 | 四川省农业科学院经济作物育种栽培研究所 | The method of seedling stage Rapid identification tobacco bred Resistance to bacterial wilt |
| CN108243891A (en) * | 2018-04-17 | 2018-07-06 | 广西壮族自治区农业科学院经济作物研究所 | A kind of disease-resistant peanut cultivation method |
Non-Patent Citations (1)
| Title |
|---|
| 花生青枯病抗性鉴定及抗源分析;周桂元 等;《花生学报》;20030930;第32卷(第3期);第25-28页 * |
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