CN110169327A - The method for building up in bacterial wilt of peanut Resistance Identification garden - Google Patents
The method for building up in bacterial wilt of peanut Resistance Identification garden Download PDFInfo
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- CN110169327A CN110169327A CN201910497711.7A CN201910497711A CN110169327A CN 110169327 A CN110169327 A CN 110169327A CN 201910497711 A CN201910497711 A CN 201910497711A CN 110169327 A CN110169327 A CN 110169327A
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- 235000020232 peanut Nutrition 0.000 title claims abstract description 75
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 20
- 241001553178 Arachis glabrata Species 0.000 title abstract 12
- 241000589771 Ralstonia solanacearum Species 0.000 claims abstract description 41
- 239000007788 liquid Substances 0.000 claims abstract description 35
- 201000010099 disease Diseases 0.000 claims abstract description 29
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 29
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- 241000894006 Bacteria Species 0.000 claims description 14
- 235000010585 Ammi visnaga Nutrition 0.000 claims description 12
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- 244000061456 Solanum tuberosum Species 0.000 claims description 10
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 239000012530 fluid Substances 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 6
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- 240000003186 Stachytarpheta cayennensis Species 0.000 claims description 3
- 235000009233 Stachytarpheta cayennensis Nutrition 0.000 claims description 3
- 239000008213 purified water Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 2
- 239000004744 fabric Substances 0.000 claims 1
- 238000012549 training Methods 0.000 claims 1
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 description 10
- 244000052769 pathogen Species 0.000 description 8
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- 244000039328 opportunistic pathogen Species 0.000 description 2
- 235000011962 puddings Nutrition 0.000 description 2
- 229930195730 Aflatoxin Natural products 0.000 description 1
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- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000146384 Glaux Species 0.000 description 1
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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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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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 kind of method for building up in bacterial wilt of peanut Resistance Identification garden, and first separation and Extraction and culture Ralstonia solanacearum liquid from bacterial wilt of peanut strain, are 3~5 × 10 with concentration7The Ralstonia solanacearum liquid of cfu/ml impregnates susceptible peanut seed, and seed soaking amount is 10 peanut seed/5ml, plants after the 30min that soaks seed；Ralstonia solanacearum liquid is sprayed at peanut blade face after peanut emergence, sprinkling in every 10 days is primary, and injures blade and be inoculated with ralstonia solanacearum again；It crushes to apply after pulling out because of the withered peanut plant of bacterial wilt evil and is sprinkled upon field, peanut in the ranks and dead seedling vacant lot plant again through seed soaking infection ralstonia solanacearum susceptible peanut seed, and continues to hurt leaf inoculation, dead seedling after emergence, reseed until harvest, diseased plant crush returning to the field again；Annual spring and autumn planted for two seasons, and dead seedling is reseeded 2~4 times, and the above steps are repeated 2~3 years, formed bacterial wilt of peanut Resistance Identification garden.Solve peanut field anti-disease enzyme garden establish the period it is long, time-consuming and laborious the problems such as.
Description
Technical field
The invention belongs to Resistance to bacterial wilt to identify field, be related to a kind of method for building up in bacterial wilt of peanut Resistance Identification garden.
Background technique
Bacterial wilt is one of the important disease in south China peanut producing region, and crop field disease incidence is generally 10%~30%, sternly
Reach 50% or more when weight.In addition to causing the underproduction, also seed nutritional quality, aflatoxin contamination etc. are had a major impact.Choosing
Educating resistance to bacterial wilt kind is to improve the most effectual way of peanut yield, and it is breeding material that breeding for disease resistance, which needs Resistant germplasm, and is showed
Some resource disease identification and selection technologies rely primarily on field Disease garden identification, and field Infected Field is because of uneven and hair of falling ill
The reasons such as sick rate is not high, it is long to lead to establish the period, time-consuming and laborious.Therefore, a kind of quick, effective, stable bacterial wilt garden is explored
Method for building up is necessary.
Summary of the invention
The purpose of the present invention is to provide a kind of method for building up in bacterial wilt of peanut Resistance Identification garden, to solve peanut field
Period length, time-consuming and laborious problem are established in anti-disease enzyme garden.
The technical scheme adopted by the invention is that the method for building up in bacterial wilt of peanut Resistance Identification garden, the specific steps are as follows:
Step S1, the separation and Extraction and culture of Ralstonia solanacearum liquid；
Step S2, Ralstonia solanacearum seed soaking plantation: susceptible peanut seed is impregnated with Ralstonia solanacearum liquid, is planted after the completion of seed soaking；
Step S3, hurt leaf inoculation: Ralstonia solanacearum liquid being sprayed at peanut blade face after peanut emergence, and injures blade and is inoculated with again
Ralstonia solanacearum；
Step S4, dead seedling is reseeded: will occur to crush to apply after the withered peanut dead seedling of bacterial wilt evil is pulled out to be sprinkled upon field, in flower
It is raw in the ranks and the susceptible peanut seed through seed soaking infection ralstonia solanacearum is planted in dead seedling vacant lot again, and continue after emergence to hurt leaf and connect
Kind, is reseeded at dead seedling, until harvest, diseased plant crushes returning to the field again；
Step S5, annual spring and autumn planted for two seasons, and the above steps are repeated 2~3 years, forms peanut resistance to bacterial wilt and identifies garden.
Further, the additional amount of the step S2 Ralstonia solanacearum liquid is 10 peanut seed/5ml.
Further in the Ralstonia solanacearum liquid concentration of the step S2 and step S4 are 3-5 × 107cfu/ml；The step
The seed soaking time of S2 is 30min.
Further, the step S3 hurts leaf inoculation and carries out once for every 10 days；Annual spring and autumn plantation two in the step S5
Season, annual dead seedling are reseeded carry out 2~4 times.
Further, the separation and Extraction of the step S1 Ralstonia solanacearum liquid realizes process are as follows: from bacterial wilt of peanut garden, discovery is opened
Begin 40~60 days seedling age peanut plants of wilting occur, main root shows as rat-tail shape, and for the tip of a root at water soaking mode, lateral root is undeveloped, in nothing
Flame sterilizes after impregnating the peanut plant root of wilting in alcohol in collarium border, cuts at butt 1/3 in main root
Disconnected, plant is standing and soaking 3~5min in aqua sterilisa downwards by notch, is observed that the bacterial ooze of white is overflowed from incision, is obtained
Ralstonia solanacearum liquid is simultaneously cultivated.
Further, the specific steps of the step S1 culture Ralstonia solanacearum liquid are as follows:
Step S11, solid medium and fluid nutrient medium are prepared: potato decortication being sliced, potato 60g, 10g, sugarcane are weighed
10g potato is diced and boils softening addition 10g sucrose, 7.5g agar, is settled to 500ml, mistake with water by sugared 60g, 10g, agar 7.5g
Filter obtains solid medium；60g potato is diced to be put into 3L water and boils softening addition sucrose 60g, 3L is settled to, liquid is obtained by filtration
State culture medium, and at 121 DEG C be placed on solid medium and fluid nutrient medium sterilizing 25min stored refrigerated in refrigerator；
Step S12, TTC solution is prepared；The 2 of 0.1g are weighed, 3,5- triphenyltetrazolium chlorides are determined with after dissolved in purified water
Hold to 10ml, TTC solution is made, and shading refrigerates；
Step S13, step S13, will will consolidate after solid medium 2 and liquid culture medium 9 take out and sterilize two hours
State culture medium, which is put into baking oven, to be kept the temperature, and culture dish is dismantled, and successively pours into solid medium, solid medium in 6 culture dishes
Height is culture dish ware inner height half, and 6.5ml TTC solution is added in remaining solid medium and shakes up, then
It successively pours into 6 culture dishes, after to be solidified, is stained with toothpick after previously extracting isolated bacterial wilt original bacteria liquid on culture dish
It gently crosses, seals culture dish with sealing adhesive plaster, and culture dish is put upside down into 28 DEG C of incubators 2 days；Occurs powder in culture dish
When red single bacterium, fluid nutrient medium is poured into centrifuge tube, with being put into centrifuge tube after toothpick picking colony, centrifuge tube is put
Enter in the shaking table that revolving speed is 150r/min, temperature is 28 DEG C, carries out the culture of Ralstonia solanacearum liquid.
Further, the step S5 forms the anti-disease enzyme garden that susceptible variety disease incidence is greater than 90%.
The invention has the advantages that quickly establishing bacterial wilt of peanut anti-disease enzyme garden.Conventional peanut field anti-disease enzyme
The foundation in garden is to accumulate continuous cropping plot pathogen, and then form serious anti-disease enzyme of falling ill
Garden, establishing the period needs 5-6；The present invention by soak seed, hurt leaf, reseed, the continuous circulation inoculation blueness of modes such as diseased plant returning to the field it is withered
Pathogen can be obviously shortened bacterial wilt of peanut anti-disease enzyme garden so that Peanut continuous cropping field bacterial wilt opportunistic pathogen doubles breeding accumulation
Establish the period, establishing anti-disease enzyme garden only needs 2~3 years, and formed susceptible variety disease incidence reach 90% or more it is anti-
It is long, time-consuming and laborious to solve the problems, such as that the period is established in peanut field anti-disease enzyme garden for disease identification garden.To disease-resistant peanut varieties breeding
And the anti-disease enzyme of progeny material is of great significance.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
The method for building up in bacterial wilt of peanut Resistance Identification garden, the specific steps are as follows:
Step S1, it identifies Ralstonia solanacearum pathogenicity and culture is carried out to High pathogenicity Ralstonia solanacearum and obtain High pathogenicity Ralstonia solanacearum
Liquid.
The separation and Extraction of peanut Ralstonia solanacearum: pure water is put into taking-up (aqua sterilisa) after sterilizing 2 hours in autoclave, is prepared
Extraction for Ralstonia solanacearum liquid.The bacterial wilt of peanut strain for beginning occur wilting is taken away from peanut cultivation garden, main root shows as rat-tail
Shape, for the tip of a root at water soaking mode, lateral root is undeveloped.The peanut plant here that withers for adopting back is cut off into branches and leaves and palpus.Be ready to sterile water,
Centrifuge tube, scissors, marker, tweezers, cotton, 75% alcohol are taken away super-clean bench disinfection by ultraviolet light 30 minutes.Material is put into super
Net platform wipes both hands in cotton intrusion alcohol.Diseased plant root is slightly impregnated in alcohol in superclean bench, flame
Incineration is cut in main root from butt 1/3, and plant is soaked in the test tube for containing aqua sterilisa (40~60 days by notch downward
1~2 plant/2ml of seedling age liquid), after standing 3~5min, observe that the bacterial ooze of white is overflowed from incision.
It prepares solid-state and liquid culture medium: potato decortication being sliced, potato 60g, 10g, sucrose 60g, 10g, agar are weighed
7.5g.10g small pudding is boiled into softening, 10g sucrose, 7.5g agar is added, is settled to 500ml with water, be put into conical flask after filtering
In, obtain solid medium.60g small pudding is put into the pot for filling 3L water and boils softening addition sucrose 60g, is settled to 3L, mistake
It filters in conical flask, obtains liquid culture medium, and 25min that the two sterilized in 121 DEG C of autoclaves is placed in refrigerator and refrigerates
It saves.
It configures TTC solution: preparing TTC solution (color developing agent) 10ml, weigh TTC (2,3,5- triphenyltetrazolium chloride)
0.1g is fitted into centrifuge tube and (protects convenient for shading) with a small amount of dissolved in purified water and with volumetric flask constant volume to 10ml, is taken with liquid-transfering gun
Pure water pours into centrifuge tube, and TTC solution need to be wrapped up with masking foil and is put into refrigerator, and shading is protected with filtering in superclean bench
It deposits.Pathogen can be displayed in red in TTC, be counted with distinguishing.
The culture of ralstonia solanacearum: solid medium 2, liquid culture medium 9 are put into autoclave from taking out in refrigerator
Sterilizing is taken out after two hours, and solid medium is put into baking oven and is kept the temperature.By alcolhol burner, culture dish, glass long tube, toothpick, shifting
Liquid rifle, two box of pipette tips are put into superclean bench ultraviolet sterilization 30min.After superclean bench disinfection, solid medium is taken to be put into
In superclean bench, after disinfecting both hands in alcohol, alcolhol burner is lighted, dismantles culture dish in superclean bench.By solid state rheology
Base successively pours into 6 culture dishes, is highly ware inner height half.The remaining culture medium in solid medium conical flask
Middle addition 6.5ml TTC solution simultaneously shakes up, and then successively pours into 6 culture dishes, after to be solidified, is stained with previous extraction with toothpick
Gently cross on culture dish after isolated bacterial wilt original bacteria liquid, it is unavailable energetically, to prevent puncturing culture medium.With sealing after pulling
Adhesive plaster seals culture dish, and adhesive plaster will be connected to each other from beginning to end, be entered with antiforeign bacteria, culture dish put upside down, to prevent water steam from up running,
Desktop is tidied up, super-clean bench is closed.It is put into 28 DEG C of incubators in 2 days or so culture dishes and pink single bacterium colony occurs, by liquid
Culture medium pours into centrifuge tube, about 2/3 amount, and centrifuge tube and culture dish are put on one side, and alcolhol burner is placed in another side after lighting,
A toothpick is clamped with tweezers, after sterilizing on alcolhol burner, opens culture dish, with being put into centrifuge tube after toothpick picking colony,
Complete needed for amount after centrifuge tube is put into shaking table, shaking speed 150r/min, temperature be 28 DEG C, pays attention to must stick tightly to prevent
It falls.After about one day, toothpick is indistinct in centrifuge tube, then bacterial concentration reaches requirement.The effect of fluid nutrient medium is to allow
Bacterium colony in solid culture growth and breeding in a liquid, it is therefore an objective to increase colony counts, liquid convenient for seed soaking infecting peanut seed,
Just be put into toothpick be in bacterium solution it is clearly visible, with the extension of incubation time, germ is increased, and bacterium solution is muddy, if making toothpick
If hidden existing, toothpick does not see Chu when germ is increased.
Step S2, Ralstonia solanacearum seed soaking plantation: by High pathogenicity Ralstonia solanacearum liquid about with 10 peanut/5ml additional amounts,
Enter and impregnate 30min in 36 peanut species of sweet osmanthus of preparation, peanut is taken out after the completion of seed soaking and is planted.It cannot be exposed to the sun under the sun,
Bacterial wilt opportunistic pathogen can be killed by being exposed to the sun.
Ralstonia solanacearum liquid concentration is about to contain Ralstonia solanacearum 3 × 10 in every milliliter of bacterium solution7~5 × 107It is a.
Using 3 × 107~5 × 107The Ralstonia solanacearum liquid immersion peanut seed 30min of cfu/ml, bacterial wilt disease incidence highest,
Disease time is most short, is conducive to the quick foundation in anti-disease enzyme garden.
Step S3, bacterial wilt bacterium solution is sprayed at peanut blade face after peanut emergence, and injures blade be inoculated with blueness again withered
Germ, inoculation in every 10 days are primary.
Seed soaking is infected from kind of portion before plantation, and injuring blade is leaves infected, and the inoculation of bacterial wilt bacterium solution is sprayed after emergence, is
In order to carry out double infection to peanut, peanut disease incidence is aggravated, and then increase the pathogen on peanut cultivation ground.
Step S4, it is withered that bacterial wilt evil occurs after peanut infection pathogen, withered dead seedling is pulled out, crushing is sprinkled upon field,
And peanut in the ranks and dead seedling vacant lot plant again again through seed soaking infection ralstonia solanacearum seed, hurt leaf inoculation, dead seedling.
The plantation of current year this season is innovative point of the invention, plants the kind of infection germ after dead seedling again again in dead seedling vacant lot
Son is equivalent to and reseeds, and withered dead seedling is pulled out, and crushing is sprinkled upon field, infects more pathogens, infection cycle with allowing this block
Germ, it is more preferable to the causative effect of peanut.
Step S5, the above steps are repeated 2~3 years, and annual two season of spring and autumn plantation peanut, dead seedling reseeds 2~4 times, most end form
Garden is identified at peanut resistance to bacterial wilt.
A situation arises compares that (compare does not include soaking for test result and normal peanut continuous cropping tibet milkwort root bacterial wilt evil
Kind, be inoculated with pathogen and recycle plantation step after hurting leaf and dead seedling, only conventional to finish harvest, the second season plants again, with normal
Advise it is different, be routinely 4~5 months harvest after plant again, planted again after method dead seedling of the invention about 2 months plantation one
It is secondary, i.e., it can be reseeded 2~4 times in spring or autumn, primary withered seedling, i.e., the dead seedling of progress in every 1~2 month occurs within 1~2 month
It reseeds.
Conventional field anti-disease enzyme garden is conventional seed planting, circulation plantation step after not soaking seed and be inoculated with pathogen, hurting leaf and dead seedling
Suddenly, it is planted again within 5 months after mature harvest, south could form the serious sick nursery of morbidity for about 5~6 years.Leaching before present invention plantation
Kind, bacterial wilt bacterium solution is sprayed at peanut blade face after emergence, and injure blade and be inoculated with ralstonia solanacearum, inoculation one in every 10 days again
Secondary, dead seedling of rooting out after dead seedling is simultaneously reseeded, and it is primary to plant plantation in about 2 months after dead seedling again, and dead seedling crushing is spilt
In field, then harvest.Annual two season of spring and autumn plantation peanut, 2~3 years formation susceptible variety disease incidence of circulation reach 90% or more
Anti-disease enzyme garden, as shown in table 1, natural the continuous cropping of Peanut continuous cropping sick nursery 3 years, bacterial wilt of peanut disease incidence only reached 51.3%, and
The present invention, which is inoculated with germ and reseeds continuous cropping, to be occurred sick nursery continuous cropping 3 years, and bacterial wilt of peanut disease incidence can reach 94.2%, identification effect
Fruit is significant.
A situation arises for bacterial wilt of peanut in 1 bacterial wilt of peanut garden establishment process of table
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the scope of the present invention.It is all
Any modification, equivalent replacement, improvement and so within the spirit and principles in the present invention, are all contained in protection scope of the present invention
It is interior.
Claims (7)
1. the method for building up in bacterial wilt of peanut Resistance Identification garden, which is characterized in that specific step is as follows:
Step S1, the separation and Extraction and culture of Ralstonia solanacearum liquid；
Step S2, Ralstonia solanacearum seed soaking plantation: susceptible peanut seed is impregnated with Ralstonia solanacearum liquid, is planted after the completion of seed soaking；
Step S3, hurt leaf inoculation: Ralstonia solanacearum liquid being sprayed at peanut blade face after peanut emergence, and injure blade be inoculated with blueness again withered
Germ；
Step S4, dead seedling is reseeded: will occur to crush to apply after the withered peanut dead seedling of bacterial wilt evil is pulled out to be sprinkled upon field, in peanut row
Between and dead seedling vacant lot plant the susceptible peanut seed through seed soaking infection ralstonia solanacearum again, and continue after emergence to hurt leaf inoculation,
Dead seedling is reseeded, until harvest, diseased plant crushes returning to the field again；
Step S5, annual spring and autumn planted for two seasons, and the above steps are repeated 2~3 years, forms peanut resistance to bacterial wilt and identifies garden.
2. the method for building up in bacterial wilt of peanut Resistance Identification according to claim 1 garden, which is characterized in that the step S2
The additional amount of Ralstonia solanacearum liquid is 10 peanut seed/5ml.
3. the method for building up in bacterial wilt of peanut Resistance Identification according to claim 1 garden, which is characterized in that the step S2
Ralstonia solanacearum liquid concentration with step S4 is 3~5 × 107cfu/ml；
The seed soaking time of the step S2 is 30min.
4. the method for building up in described in any item bacterial wilt of peanut Resistance Identifications garden according to claim 1~3, which is characterized in that
The step S3 hurts leaf and is inoculated with progress in every 10 days once；
Annual spring and autumn planted for two seasons in the step S5, and annual dead seedling is reseeded carry out 2~4 times.
5. the method for building up in bacterial wilt of peanut Resistance Identification according to claim 4 garden, which is characterized in that the step S1
The separation and Extraction of Ralstonia solanacearum liquid realizes process are as follows: the flower that discovery is withered by bacterial wilt of peanut from the peanut field of 40~60 days seedling ages
Raw plant, main root show as rat-tail shape, and for the tip of a root at water soaking mode, lateral root is undeveloped, by the peanut plant of wilting in gnotobasis
Flame sterilizes after root is impregnated in alcohol, cuts at butt 1/3 in main root, plant is standing and soaking downwards by notch
3~5min in aqua sterilisa observes that the bacterial ooze of white is overflowed from incision, obtains Ralstonia solanacearum liquid and cultivated.
6. the method for building up in bacterial wilt of peanut Resistance Identification according to claim 5 garden, which is characterized in that the step S1
Cultivate the specific steps of Ralstonia solanacearum liquid are as follows:
Step S11, solid medium and fluid nutrient medium are prepared: potato decortication being sliced, potato 60g, 10g, sucrose are weighed
10g potato is diced and boils softening addition 10g sucrose, 7.5g agar, is settled to 500ml with water, filter by 60g, 10g, agar 7.5g
Obtain solid medium；60g potato is diced to be put into 3L water and boils softening addition sucrose 60g, 3L is settled to, liquid is obtained by filtration
Culture medium, and at 121 DEG C be placed on solid medium and fluid nutrient medium sterilizing 25min stored refrigerated in refrigerator；
Step S12, TTC solution is prepared；Weigh the 2 of 0.1g, 3,5- triphenyltetrazolium chlorides, with being settled to after dissolved in purified water
TTC solution is made in 10ml, and shading refrigerates；
Step S13, after solid medium 2 and liquid culture medium 9 being taken out and sterilized two hours, solid medium is put into
It is kept the temperature in baking oven, dismantles culture dish, solid medium is successively poured into 6 culture dishes, solid medium height is culture dish
Inner height half, and 6.5ml TTC solution is added in remaining solid medium and shakes up, then successively pour into 6 trainings
It supports in ware, after to be solidified, be stained with toothpick after previously extracting isolated bacterial wilt original bacteria liquid and gently crossed on culture dish, use is close
Sealing cloth seals culture dish, and culture dish is put upside down into 28 DEG C of incubators 2 days；When occurring pink single bacterium in culture dish,
Fluid nutrient medium is poured into centrifuge tube, with being put into centrifuge tube after toothpick picking colony, it is 150r/ that centrifuge tube, which is put into revolving speed,
In min, the shaking table that temperature is 28 DEG C, the culture of Ralstonia solanacearum liquid is carried out.
7. the according to claim 1, method for building up in bacterial wilt of peanut Resistance Identification garden described in 2,3,5 or 6, which is characterized in that
The step S5 forms the anti-disease enzyme garden that susceptible variety disease incidence is greater than 90%.
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Application publication date: 20190827 Assignee: Guangxi Nanning Lvshan Agricultural Technology Co.,Ltd. Assignor: GUANGXI ZHUANG AUTONOMOUS REGION ACADEMY OF AGRICULTURAL SCIENCES Contract record no.: X2023980046294 Denomination of invention: Establishment Method of Peanut Bacterial Wilt Resistance Identification Nursery Granted publication date: 20210727 License type: Common License Record date: 20231108 |