CN109566373A - A method of inoculation study bacterial wilt is impregnated by root - Google Patents
A method of inoculation study bacterial wilt is impregnated by root Download PDFInfo
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- CN109566373A CN109566373A CN201811606513.1A CN201811606513A CN109566373A CN 109566373 A CN109566373 A CN 109566373A CN 201811606513 A CN201811606513 A CN 201811606513A CN 109566373 A CN109566373 A CN 109566373A
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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
- A01G31/00—Soilless cultivation, e.g. hydroponics
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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
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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Abstract
The present invention provides a kind of methods that inoculation study bacterial wilt is impregnated by root, belong to crop pest management technical field.The method provided by the invention that inoculation study bacterial wilt is impregnated by root is inoculated in the culture medium of culture tissue-cultured seedling by simulating soil setting in culture vessel, and by germ, is infected tissue-cultured seedling by inoculation germ, is studied causing a disease for bacterial wilt；Easy to operate, workload is small, saves the time of experiment；With application value.
Description
Technical field
The present invention relates to crop pest management technical fields, are ground in particular to a kind of by root dipping inoculation
Study carefully the method for bacterial wilt.
Background technique
Bacterial wilt is the bacillary soil-borne disease as caused by Ralstonia solanacearum (Ralstoniasolanacearum), energy
250 various plants for infecting 54 sections, including some important crops and industrial crops, as potato, tomato, tobacco,
Eggplant etc..The bacterium is a kind of Gram negative rods bacterium of aerobic-type, and optimum growth temperature is 28 DEG C -35 DEG C, is had very
Strong survival ability and adaptability can survive in water, soil and plant residue, can also perceive the secretion of plant root
Liquid, and carry out root using travelling flagellum and infect.Under usual conditions, Ralstonia solanacearum infects plant by wound and natural aperture
Object, just Ralstonia solanacearum is colonized in root cortex when invasion, is spread after reaching skeleton by xylem vessel, sharp in pin main body
Massive duplication is carried out after being colonized with the completion of the nutriment of plant itself, thus cause the generation of virulence factor and exocellular polysaccharide,
Cause the destruction and the final wilting of plant of fibrovascular system.
In the conventional method of Ralstonia solanacearum Study on Pathogenicity, either it is inoculated with from root or is inoculated with from cauline leaf,
It is required to through potting culture plant, which not only needs certain growth cycle, and time-consuming, and in plant incubation
The series of steps such as ridging, carrying, watering need to consume a large amount of manpower and material resources, while also needing to occupy the culture of large area
Frame is unfavorable for effectively pushing for scientific experiment.In addition, cauline leaf inoculation with bacterium infect under field conditions (factors) plant mode difference compared with
Greatly, experiment effect is unsatisfactory, and conventional root inoculation needs first to hurt root and could be inoculated with, and process is cumbersome, and utilizes soil incubation
Cause Ralstonia solanacearum effect of inoculation unstable because soil block is unevenly distributed after plant.
Summary of the invention
The object of the present invention is to provide a kind of methods that inoculation study bacterial wilt is impregnated by root, are soaked by root
The method of stain very easily realizes the research to crop bacterial wilt.
In order to realize above-mentioned purpose of the invention, using following technical scheme:
A method of inoculation study bacterial wilt is impregnated by root, comprising the following steps:
Water planting tissue cultural seedlings of free, and culture of rootage is carried out, obtain water planting plant；
The root of water planting plant is immersed in the lighttight culture vessel added with culture medium and is cultivated, group is obtained
Cultivate strain；
And Ralstonia solanacearum liquid is introduced into inoculation dipping culture tissue culture plant in culture vessel；
Continue to cultivate and carry out the disease survey of plant after inoculation Ralstonia solanacearum liquid.
Compared with prior art, the beneficial effect comprise that provided by the invention impregnate inoculation study by root
The method of bacterial wilt is inoculated in the culture medium of culture tissue-cultured seedling by simulating soil setting in culture dish, and by germ, leads to
Inoculation germ infection tissue-cultured seedling is crossed, causing a disease for bacterial wilt is studied；Easy to operate, workload is small, saves the time of experiment；With pushing away
Wide application value.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
A kind of method for impregnating inoculation study bacterial wilt by root of the embodiment of the present invention is specifically described below.
A method of inoculation study bacterial wilt is impregnated by root, comprising the following steps:
The root for the seedling that the root of the Rooted Cuttings obtained by tissue culture or germination obtain is immersed in lighttight addition
It is cultivated in the culture vessel of nutritious liquid, obtains water planting plant；
And Ralstonia solanacearum liquid is introduced into the culture vessel added with nutrient solution；Dipping culture water planting plant；
Continue to cultivate and carry out the disease survey of plant after inoculation Ralstonia solanacearum liquid.
The tissue-cultured seedling for selecting detoxification is after the tissue-cultured seedling based on detoxification is avoided that plant virus infection, due to the life of virus
Length is also required to nutrient, will keep competitive relation with plant, consume nutrient, slow down the growth of plant, even result in plant and die of illness.
Using the tissue-cultured seedling of detoxification, and the interference of the other viruses of discharge, shadow of the more accurate research bacterial wilt to plant pathogenic
It rings.Using the method for previous soil incubation, the virus of soil easily causes interference.
In addition, selecting the growing environment of lighttight culture vessel in the soil convenient for simulation plant, while can also prevent
Algal grown maintains the stabilization of the nutrient environment of plant strain growth.
In some optional embodiments of the present invention, Rooted Cuttings after the clipped culture to 3-5 leaf of tissue-cultured seedling by transplanting, taking root
Culture obtains.
Since the differentiation capability of Rooted Cuttings is stronger, by shearing Rooted Cuttings, each position of Rooted Cuttings lives through cultivation,
With the ability for developing into whole strain, therefore by shearing tissue-cultured seedling, the amount for obtaining plant can be expanded.
In some optional embodiments of the present invention, tissue cultural seedlings of free obtains tissue-cultured seedling through culture medium culture to 3-5 leaf age
Or the temperature of culture seedling is 15-20 DEG C, relative air humidity 67%-75%.
Detoxification treatment tissue-cultured seedling avoids experiment disturbed, improves the reliability of experiment.The plant studied will be needed to take off
Processing the methods of is broken up in differentiation again；Tissue-cultured seedling is obtained by callus tissue culture, then by tissue-cultured seedling detoxification, group can be kept
The growth for training seedling is consistent, process control.
Due to Initial stage of culture, the ability for resisting extraneous influence of tissue-cultured seedling is weak, needs a mild condition of culture, therefore
15-20 DEG C, relative air humidity is cultivated under conditions of being 67%-75%, can guarantee that tissue-cultured seedling stablizes growth.
Due to Initial stage of culture, the ability for resisting extraneous influence of tissue-cultured seedling is weak, after tissue-cultured seedling detoxification, culture to 3-5 leaf
Root media is added in age, carries out culture of rootage.
By culture of rootage, it is that tissue-cultured seedling generates root, can be just engaged in subsequent inoculation work, just can be carried out connecing for Ralstonia solanacearum
Kind.
In some optional embodiments of the present invention, the time of culture of rootage is 25-33 days.
In some optional embodiments of the present invention, Ralstonia solanacearum liquid is that liquid training is inoculated in by picking Ralstonia solanacearum single colonie
Base is supported, and cultivates and obtains under the conditions of 25-30 DEG C.
In some optional embodiments of the present invention, tissue-cultured seedling is tissue culture seedlings of potatoes, Tissue-cultured Tomato Plants, eggplant tissue-cultured seedling
With one kind in tobacco tissue-cultured seedling；Seedling is germinateed by one of tomato seeds, eggplant seed, pepper seed or tobacco seed to be cultivated
It obtains.
By the infection research of a variety of tissue-cultured seedling, it can more comprehensively be apparent from infection and the pathogenesis of bacterial wilt.
By the culture of suitable time, tissue-cultured seedling generates root, and when being inoculated with Ralstonia solanacearum liquid, Ralstonia solanacearum could more
It is easy dip dyeing, the more easily research of the mechanism of catching an illness of research bacterial wilt.
In some optional embodiments of the present invention, the OD of Ralstonia solanacearum liquid600For 0.08-0.11.
The excessive concentration of bacterium solution is easy to cause plant to infect excessive Ralstonia solanacearum, due to the general resilience of tissue-cultured seedling compared with
It is weak, it is easy to cause plant dead.And gelled fluid concentration is low, and effect is not achieved, and the long period is needed to can be only achieved experiment effect；
Experimentation and time are extended, nor being suitble to；Therefore the OD of selected Ralstonia solanacearum liquid600For 0.08-0.11.
In some optional embodiments of the present invention, Ralstonia solanacearum single colonie is inoculated in BGT plate by Ralstonia solanacearum and is activated
Culture obtains；BGT plate includes peptone 10g/L, glucose 2.5g/L, acid hydrolyzed casein 1g/L, agar powder 15g/L and 2,
3,5- triphenyltetrazolium chloride 0.05g/L.
By the method for plate streaking, Isolates of Pseudomonas Solanacearum Smith is activated, is rejuvenated, facilitates and continues to cultivate.
In some optional embodiments of the present invention, Ralstonia solanacearum liquid is introduced into the introduction volume and culture vessel in culture dish
The volume ratio of culture solution is 1:30-40.
The introduction volume of the Ralstonia solanacearum liquid of proper ratio, can guarantee the normal growth of plant, can also maintain the infection of Ralstonia solanacearum.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of methods that inoculation study bacterial wilt is impregnated by root, in the present embodiment, with potato
The root for carrying out Ralstonia solanacearum as target crop impregnates inoculation study, comprising the following steps:
The Potato Cultivars that 1.1 selections need to test, culture obtains tissue culture seedlings of potatoes in MS culture medium；
1.2 pairs of tissue culture seedlings of potatoes carry out detoxification treatment, obtain potato detoxicating tissue-cultured seedling；
1.3 cultivate potato detoxicating tissue-cultured seedling under conditions of 15 DEG C and relative air humidity are 65%, and one week or so
3-5 leaf age is grown to potato detoxicating tissue-cultured seedling；
1.4 pairs of potato detoxicating tissue-cultured seedling continue to cultivate after shearing；
The 1.5 potato detoxicating tissue-cultured seedling after shearing grows to 3-5 leaf age, then carries out water planting and cuttage transplanting, adds
Enter root media to carry out culture of rootage 33 days, obtains water planting plant；
Water planting plant is placed into the lighttight culture vessel added with Hoagland culture medium (in the present embodiment by 1.6
Preferably glass tube) in, and it is made into buoy with plastic foam, osculum is cut off along buoy radius, water planting plant is clamped in osculum
In；
Culture vessel is cultivated 16h by 1.7 under the conditions of 25 DEG C of temperature, and foundation actual conditions add culture medium；
Ralstonia solanacearum is passed through plate streaking method by 1.8, and being inoculated in BGT plate, (BGT plating medium includes peptone
10g/L, glucose 2.5g/L, acid hydrolyzed casein 1g/L, agar powder 15g/L and 2,3,5- triphenyltetrazolium chloride 0.05g/
L), cultivated at 32 DEG C；
Picking edge milky, intermediate pink and more liquid single colonie carry out 32 DEG C after 1.9 cultures 2-4 days
Liquid Culture is configured to OD600For 0.08 Ralstonia solanacearum liquid；
1.10 hold the culture added with Hoagland culture medium that Ralstonia solanacearum liquid is added to culture water culture of potato plant
In device, the volume ratio of Ralstonia solanacearum liquid and culture medium is 1:30.
Start to observe plant after 1.11 inoculation Ralstonia solanacearum liquid, plant carries out disease survey after merging.
It is divided into 5 morbidity grades according to plant wilt degree: 0 grade=without wilting symptom, 1 grade=1%-25% blade withers
It is listless；2 grades=26%-50% blade is wilted；3 grades=51%-75% blade is wilted；4 grades=76%-100%, calculate the hair of plant
Sick index D I=(n1X1+n2X2+n3X3+n4X4)/(n0+n1+n2+n3+n4) X4, n0, n1, n2, n3, n4 represent morbidity grade
Respectively 0,1,2,3,4 number, DI=0 is represented without wilting (No Wilted Symptom, N)；DI=0.1-1.5 represents anti-
Disease (Resistant, R) DI=1.6-3.0 resists (Medium Resistant, MR) in representing；DI=3.1-4.0 represents susceptible
(Susceptible, S).
Embodiment 2
The present embodiment provides a kind of methods that inoculation study bacterial wilt is impregnated by root, in the present embodiment, are made with tomato
The root for carrying out Ralstonia solanacearum for target crop impregnates inoculation study, comprising the following steps:
The tomato varieties that 1.1 selections need to test, germinate tomato seeds, obtains tomato seedling, takes the tip of a root of tomato seedling, general kind
The eggplant seedling tip of a root is cultivated in MS culture medium and obtains Tissue-cultured Tomato Plants；
1.2 pairs of Tissue-cultured Tomato Plants carry out detoxification treatment, obtain tomato tissue cultural seedlings of free；
1.3 cultivate tomato tissue cultural seedlings of free under conditions of 20 DEG C and relative air humidity are 75%, and one week or so extremely
Tomato tissue cultural seedlings of free grows to 3-5 leaf age；
1.4 pairs of tomato tissue cultural seedlings of free continue to cultivate after shearing；
The 1.5 tomato tissue cultural seedlings of free after shearing grows to 3-5 leaf age, then carries out water planting and cuttage transplanting, is added
Root media carries out culture of rootage 25 days, obtains water planting plant；
Water planting plant is placed into the lighttight culture vessel added with Hoagland culture medium (in the present embodiment by 1.6
Preferably glass tube) in, and it is made into buoy with plastic foam, osculum is cut off along buoy radius, water planting plant is clamped in osculum
In；
Culture vessel is cultivated 16h by 1.7 under the conditions of 25 DEG C of temperature, and foundation actual conditions add culture medium；
1.8 being inoculated in BGT plate, (BGT plating medium includes peptone by Ralstonia solanacearum by plate streaking method
10g/L, glucose 2.5g/L, acid hydrolyzed casein 1g/L, agar powder 15g/L and 2,3,5- triphenyltetrazolium chloride 0.05g/
L), cultivated at 25 DEG C；
Picking edge milky, intermediate pink and more liquid single colonie carry out 25 DEG C after 1.9 cultures 2-4 days
Liquid Culture is configured to OD600For 0.11 Ralstonia solanacearum liquid；
1.10 are added to Ralstonia solanacearum liquid the culture vessel added with Hoagland culture medium that culture tomato juice cultivates strain
Interior, the volume ratio of Ralstonia solanacearum liquid and culture medium is 1:40.
Start to observe plant after 1.11 inoculation Ralstonia solanacearum liquid, plant carries out disease survey after merging.
It is divided into 5 morbidity grades according to plant wilt degree: 0 grade=without wilting symptom, 1 grade=1%-25% blade withers
It is listless；2 grades=26%-50% blade is wilted；3 grades=51%-75% blade is wilted；4 grades=76%-100%, calculate the hair of plant
Sick index D I=(n1X1+n2X2+n3X3+n4X4)/(n0+n1+n2+n3+n4) X4, n0, n1, n2, n3, n4 represent morbidity grade
Respectively 0,1,2,3,4 number, DI=0 is represented without wilting (No Wilted Symptom, N)；DI=0.1-1.5 represents anti-
Disease (Resistant, R) DI=1.6-3.0 resists (Medium Resistant, MR) in representing；DI=3.1-4.0 represents susceptible
(Susceptible, S).
Embodiment 3
The present embodiment provides a kind of methods that inoculation study bacterial wilt is impregnated by root, in the present embodiment, are made with tobacco
The root for carrying out Ralstonia solanacearum for target crop impregnates inoculation study, comprising the following steps:
The tobacco bred that 1.1 selections need to test, germinate tobacco seed, obtains tobacco seedling, takes the tip of a root of tobacco seedling, by cigarette
The careless seedling tip of a root is cultivated in MS culture medium and obtains tobacco tissue-cultured seedling；
1.2 pairs of tobacco tissue-cultured seedling carry out detoxification treatment, obtain tobacco tissue cultural seedlings of free；
1.3 cultivate tobacco tissue cultural seedlings of free under conditions of 18 DEG C and relative air humidity are 70%, and one week or so extremely
Tobacco tissue cultural seedlings of free grows to 3-5 leaf age；
1.4 pairs of tobacco tissue cultural seedlings of free continue to cultivate after shearing；
The 1.5 tobacco tissue cultural seedlings of free after shearing grows to 3-5 leaf age, then carries out water planting and cuttage transplanting, is added
Root media carries out culture of rootage 30 days, obtains water planting plant；
Water planting plant is placed into the lighttight culture vessel added with Hoagland culture medium (in the present embodiment by 1.6
Preferably glass tube) in, and it is made into buoy with plastic foam, osculum is cut off along buoy radius, water planting plant is clamped in osculum
In；
Culture vessel is cultivated 16h by 1.7 under the conditions of 25 DEG C of temperature, and foundation actual conditions add culture medium；
1.8 being inoculated in BGT plate, (BGT plating medium includes peptone by Ralstonia solanacearum by plate streaking method
10g/L, glucose 2.5g/L, acid hydrolyzed casein 1g/L, agar powder 15g/L and 2,3,5- triphenyltetrazolium chloride 0.05g/
L), cultivated at 28 DEG C；
Picking edge milky, intermediate pink and more liquid single colonie carry out 28 DEG C after 1.9 cultures 2-4 days
Liquid Culture is configured to OD600For 0.1 Ralstonia solanacearum liquid；
1.10 are added to Ralstonia solanacearum liquid the culture vessel added with Hoagland culture medium that culture tabacco water cultivates strain
Interior, the volume ratio of Ralstonia solanacearum liquid and culture medium is 1:35.
Start to observe plant after 1.11 inoculation Ralstonia solanacearum liquid, plant carries out disease survey after merging.
It is divided into 5 morbidity grades according to plant wilt degree: 0 grade=without wilting symptom, 1 grade=1%-25% blade withers
It is listless；2 grades=26%-50% blade is wilted；3 grades=51%-75% blade is wilted；4 grades=76%-100%, calculate the hair of plant
Sick index D I=(n1X1+n2X2+n3X3+n4X4)/(n0+n1+n2+n3+n4) X4, n0, n1, n2, n3, n4 represent morbidity grade
Respectively 0,1,2,3,4 number, DI=0 is represented without wilting (No Wilted Symptom, N)；DI=0.1-1.5 represents anti-
Disease (Resistant, R) DI=1.6-3.0 resists (Medium Resistant, MR) in representing；DI=3.1-4.0 represents susceptible
(Susceptible, S).
Embodiment 4
The present embodiment provides a kind of methods that inoculation study bacterial wilt is impregnated by root, in the present embodiment, are made with eggplant
The root for carrying out Ralstonia solanacearum for target crop impregnates inoculation study, comprising the following steps:
The Eggplant Varieties that 1.1 selections need to test, germinate eggplant seed, obtains eggplant seedling, takes the tip of a root of eggplant seedling, by eggplant
The sub- seedling tip of a root is cultivated in MS culture medium and obtains eggplant tissue-cultured seedling；
1.2 pairs of eggplant tissue-cultured seedling carry out detoxification treatment, obtain eggplant tissue cultural seedlings of free；
1.3 cultivate eggplant tissue cultural seedlings of free under conditions of 18 DEG C and relative air humidity are 68%, and one week or so extremely
Eggplant tissue cultural seedlings of free grows to 3-5 leaf age；
1.4 continuing to cultivate after shearing eggplant tissue cultural seedlings of free；
The 1.5 eggplant tissue cultural seedlings of free after shearing grows to 3-5 leaf age, then carries out water planting and cuttage transplanting, is added
Root media carries out culture of rootage 31 days, obtains water planting plant；
Water planting plant is placed into the lighttight culture vessel added with Hoagland culture medium (in the present embodiment by 1.6
Preferably glass tube) in, and it is made into buoy with plastic foam, osculum is cut off along buoy radius, water planting plant is clamped in osculum
In；
Culture vessel is cultivated 16h by 1.7 under the conditions of 25 DEG C of temperature, and foundation actual conditions add culture medium；
1.8 being inoculated in BGT plate, (BGT plating medium includes peptone by Ralstonia solanacearum by plate streaking method
10g/L, glucose 2.5g/L, acid hydrolyzed casein 1g/L, agar powder 15g/L and 2,3,5- triphenyltetrazolium chloride 0.05g/
L), cultivated at 28 DEG C；
Picking edge milky, intermediate pink and more liquid single colonie carry out 29 DEG C after 1.9 cultures 2-4 days
Liquid Culture is configured to OD600For 0.09 Ralstonia solanacearum liquid；
1.10 are added to Ralstonia solanacearum liquid the culture vessel added with Hoagland culture medium of culture eggplant water planting plant
Interior, the volume ratio of Ralstonia solanacearum liquid and culture medium is 1:38.
Start to observe plant after 1.11 inoculation Ralstonia solanacearum liquid, plant carries out disease survey after merging.
It is divided into 5 morbidity grades according to plant wilt degree: 0 grade=without wilting symptom, 1 grade=1%-25% blade withers
It is listless；2 grades=26%-50% blade is wilted；3 grades=51%-75% blade is wilted；4 grades=76%-100%, calculate the hair of plant
Sick index D I=(n1X1+n2X2+n3X3+n4X4)/(n0+n1+n2+n3+n4) X4, n0, n1, n2, n3, n4 represent morbidity grade
Respectively 0,1,2,3,4 number, DI=0 is represented without wilting (No Wilted Symptom, N)；DI=0.1-1.5 represents anti-
Disease (Resistant, R) DI=1.6-3.0 resists (Medium Resistant, MR) in representing；DI=3.1-4.0 represents susceptible
(Susceptible, S).
In conclusion the method provided in an embodiment of the present invention for impregnating inoculation study bacterial wilt by root, passes through culture
Then nontoxic tissue-cultured seedling in the culture medium of culture tissue-cultured seedling, will pass through Ralstonia solanacearum liquid inductance in the Ralstonia solanacearum liquid combination of activation
It contaminates tissue-cultured seedling and studies bacterial wilt, omit the process cultivated suddenly, save experiment flow, reduce human input, improve conventional efficient.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
1. a kind of method for impregnating inoculation study bacterial wilt by root, which comprises the following steps:
The root for the seedling that the root of the Rooted Cuttings obtained by tissue culture or germination obtain is immersed in lighttight added with battalion
It is cultivated in the culture vessel of nutrient solution, obtains water planting plant；
And Ralstonia solanacearum liquid is introduced into the nutrient solution of the culture vessel added with the nutrient solution；Dipping cultivates the water
Cultivate strain；
Continue to cultivate and carry out the disease survey of plant after being inoculated with the Ralstonia solanacearum liquid.
2. the method according to claim 1 for impregnating inoculation study bacterial wilt by root, which is characterized in that the water planting
Seedling is obtained by transplanting, culture of rootage after the clipped culture to 3-5 leaf of tissue-cultured seedling.
3. the method according to claim 2 for impregnating inoculation study bacterial wilt by root, which is characterized in that the tissue culture
Seedling is obtained by tissue cultural seedlings of free through culture medium culture to 3-5 leaf age.
4. the method according to claim 3 for impregnating inoculation study bacterial wilt by root, which is characterized in that the detoxification
The temperature that tissue-cultured seedling obtains the tissue-cultured seedling or the culture seedling through culture medium culture to 3-5 leaf age is 15-20 DEG C, air phase
It is 67%-75% to humidity.
5. the method according to claim 4 for impregnating inoculation study bacterial wilt by root, which is characterized in that described to take root
The time of culture is 25-33 days.
6. according to the described in any item methods for impregnating inoculation study bacterial wilt by root of claim 2-5, which is characterized in that
The tissue-cultured seedling is a kind of in tissue culture seedlings of potatoes, Tissue-cultured Tomato Plants, eggplant tissue-cultured seedling and tobacco tissue-cultured seedling；The seedling by kind
The germination culture of one of eggplant seed, eggplant seed, pepper seed or tobacco seed obtains.
7. the method according to claim 1 for impregnating inoculation study bacterial wilt by root, which is characterized in that the blueness is withered
Bacterium solution is to be inoculated in fluid nutrient medium by picking Ralstonia solanacearum single colonie, and cultivate and obtain under the conditions of 25-30 DEG C.
8. the method according to claim 7 for impregnating inoculation study bacterial wilt by root, which is characterized in that the blueness is withered
The OD of bacterium solution600For 0.08-0.11.
9. the method according to claim 8 for impregnating inoculation study bacterial wilt by root, which is characterized in that the blueness is withered
Bacterium single colonie is inoculated in BGT plate by Ralstonia solanacearum and activation culture obtains；The BGT plating medium includes peptone 10g/
L, glucose 2.5g/L, acid hydrolyzed casein 1g/L, agar powder 15g/L and 2,3,5- triphenyltetrazolium chloride 0.05g/L.
10. the method according to claim 9 for impregnating inoculation study bacterial wilt by root, which is characterized in that the blueness
The volume ratio that withered bacterium solution is introduced into culture solution in the introduction volume in culture dish and the culture vessel is 1:30-40.
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