CN108377893A - A kind of rapid detection method of eggplant verticillium wilt disease resistance - Google Patents
A kind of rapid detection method of eggplant verticillium wilt disease resistance Download PDFInfo
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- CN108377893A CN108377893A CN201810148879.2A CN201810148879A CN108377893A CN 108377893 A CN108377893 A CN 108377893A CN 201810148879 A CN201810148879 A CN 201810148879A CN 108377893 A CN108377893 A CN 108377893A
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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
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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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 invention discloses a kind of rapid detection methods of eggplant verticillium wilt disease resistance.The method of the present invention is specific as follows：The verticillium wilt pathogenic bacteria being separated and identified are cultivated in PDA culture medium first, after it covers with plate, the eggplant seed that need to be detected is carried out disinfection successively, vernalization, and directly the seed after vernalization is sowed in the PDA culture medium for covering with verticillium wilt pathogenic bacteria, vermiculite after covering high-temperature sterilizing above, then culture medium after planting is placed in 25 DEG C of incubators and is cultivated, after cotyledon expansion, growth gesture is instituted an inquiry, its Disease resistance index and resistant are determined by the growth potential of seedling.It is experimentally confirmed：The detection method result of the present invention is accurate and easy to operate, time saving and energy saving.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of rapid detection method of eggplant verticillium wilt disease resistance.
Background technology
Verticillium wilt is a kind of common soil-borne disease caused by verticillium dahliae, and the pathogen is mainly by infecting plant
Root system blocks plant roots stem vascular bundle and in turn results in plant morbidity, and Vegetables host includes eggplant, tomato etc..Especially by
It causes verticillium wilt disease that getting worse occurs in canopy room eggplant continuous cropping, serious harm is caused to production.
Current common verticillium wilt detection method is single, mainly by spore suspension liquid irrigating root or by piercing stem, thorn root
The methods of, i.e., by preparing verticillium wilt pathogen spore suspension, to host plant progress pouring root, either basal part of stem or root are stabbed in use
The method of portion's inoculating spores suspension.Although this method extensive use, it prepares spore suspension tedious process and inoculation
Time-consuming for plant morbidity afterwards, and entire detection method takes and is 1 month or more time.In seeded process, due to inoculation soil
Earth microbial population itself and biomass have reached dynamic equilibrium, and pathogen spore is often resulted in after inoculation in the soil by soil
Have microorganism to repel, causes its time-to-live very short, it is difficult to play its causative effect.After inoculation plant disease time compared with
It is long, it is also often influenced by extraneous factor in pathogenic process, interference is generated to its detection result.
Invention content
The technical problem to be solved by the present invention is to how quickly and effectively detect eggplant verticillium wilt resistance.
In order to solve the above-mentioned technical problem.The present invention provides a kind of rapid detection methods of eggplant verticillium wilt resistance.
The rapid detection method of eggplant verticillium wilt resistance provided by the invention includes the following steps：
1) eggplant seed is seeded in the culture medium containing verticillium wilt pathogen, obtains sowing wild Oryza species；
2) matrix is covered into the seed upper layer in the sowing wild Oryza species, obtains the culture medium of covering matrix；
3) culture medium of the covering matrix is cultivated, is cultivated to the cotyledon expansion of eggplant seedling, according to seedling
Growth potential determine the resisting verticillium type of eggplant.
Above-mentioned steps 1) in, the eggplant seed also includes the steps that disinfection and vernalization successively prior to seeding.
Further, the method for the disinfection includes the following steps：By eggplant seed successively in sterile water, ethanol water
It is impregnated in liquor natrii hypochloritis, seed after being sterilized.
The disinfection can specifically carry out as follows：Eggplant seed is impregnated in sterile water first, makes the eggplant
Pathogen on seed is in the state sprouted, and then soaks eggplant seed in ethanol water and liquor natrii hypochloritis successively
Bubble kills the pathogen after activation, the seed after being sterilized.
Further, the ethanol water is the ethanol water that volume fraction is 75%；
The liquor natrii hypochloritis is the liquor natrii hypochloritis that mass concentration is 5%；
The time impregnated in the sterile water can be 1-2 hours；
The time impregnated in the ethanol water and the liquor natrii hypochloritis can be 30s, and the step is repeatable
3-5 times；
Include the steps that using aseptic water washing after the immersion；The number of the flushing can be 2-3 times.
The method of the vernalization includes the following steps：It will be placed in 25-28 with the seed after the clean disinfection of aseptic water washing
Vernalization is carried out under the conditions of DEG C.
Above-mentioned steps 1) in, the preparation method of the culture medium containing verticillium wilt pathogen includes the following steps：It will be yellow
Disease of withering pathogen is seeded in the culture dish containing PDA culture medium, and culture covers with bacterium plate to the pathogen, obtains withering containing Huang
The culture medium of sick pathogen.
Further, the condition of the culture is 25 DEG C, is protected from light culture 7-10 days；PDA culture medium in the culture dish
Thickness is at least 3mm.In the present invention, the thickness of PDA culture medium is specially 3mm in the culture dish, the rule of the culture dish
Lattice are diameter 80mm, height 10mm.
In the present invention, the verticillium wilt pathogen is the verticillium wilt pathogen by separation identification.The separation identification
The specific method is as follows：The disease of the morbidity eggplant of field acquisition is good for junction and carries out alcohol disinfecting, disinfects and is placed on nothing
It is cultivated in bacterium PDA culture medium, after it grows a large amount of mycelia, observation is taken pictures under the microscope first, then arrives mycelia tieback
After the onset of waiting for eggplant, pathogen is separately cultured according still further to the above method from morbidity place for the rhizome portion of healthy eggplant, waits growing a large amount of
Observed under the microscope after mycelia, if the observation of acquired results and early period is taken pictures result it is consistent and with the verticillium wilt pathogen reported
Form it is completely the same, then the provable pathogen be verticillium wilt pathogen.In the present invention, the verticillium wilt pathogen is specific
For verticillium dahliae Verticillium dahliae.
Above-mentioned steps 1) in, the method for sowing is uniformly to sow the eggplant seed after vernalization in each culture dish
In.In the present invention, the eggplant seed after 50 vernalization is specifically sowed in each culture dish.
Above-mentioned steps 2) in, the matrix can be mixed substrate, if vermiculite and coconut palm chaff matrix are 1 according to mass ratio：1 mixing
Obtained mixed-matrix or single-matrix, such as vermiculite.In the present invention, the matrix is specially vermiculite.
Above-mentioned steps 2) in, the thickness of the matrix is concordant with the culture dish oral area.
Above-mentioned steps 2) in, the matrix also includes the steps that sterilizing and wetting successively before covering.
Further, the condition of the sterilizing is：121 DEG C sterilize 30 minutes；The matrix is soaked using sterile water, is had
Body, the hand-holdable small watering being atomized uniformly sprays matrix.
In the above method, the step 1) and the step 2) aseptically operate.
Above-mentioned steps 3) in, the method that the culture medium of the covering matrix is cultivated is included the following steps：With sealing
Film will be cultivated after the culture medium sealing of the covering matrix, and culture to eggplant removes sealed membrane after emerging, it is made normally to open up
Leaf is grown.The temperature of the culture can be 25-28 DEG C, can not only keep pathogen active under the conditions of 25-28 DEG C, but also can protect
Card seed is normally emerged.In the present invention, the temperature of the culture is specially 25 DEG C.
Above-mentioned steps 3) in, the method that the growth potential according to seedling determines the resisting verticillium type of eggplant includes as follows
Step：Its disease index is determined according to the growth potential of seedling, its resisting verticillium type is determined further according to the disease index.It is described
The growth potential of seedling includes cotyledon stretching, extension situation, cotyledon yellow situation, hypocotyl stretching, extension situation, hypocotyl growth and infects feelings
Condition, root growth and infect situation.
Further, its disease index is determined according to the growth potential of seedling, determines that its is anti-yellowing further according to the disease index
The method of disease of withering type includes the following steps：Its state of an illness grade is determined according to the growth potential of seedling, further according to the state of an illness grade
Its disease index is calculated, its resisting verticillium type is finally determined according to the disease index；
The growth potential according to seedling determines that the standard of its state of an illness grade is as follows：
0 grade：Cotyledon stretches；Hypocotyl is healthy and strong；Root growth is good.
1 grade：Cotyledon stretches；Hypocotyl is healthy and strong；Main root growth vigor is limited, but lateral root growth is vigorous.
2 grades：Cotyledon stretching, extension is limited, aetiolation occurs；Hypocotyl growth is very thin；Main root microbiological contamination leads to dark brown, growth
Slowly, lateral root amount is few.
3 grades：Cotyledon do not stretch, yellow；Hypocotyl is very thin；Root system main root microbiological contamination leads to dark brown, slow-growing, has a small amount of
Lateral root generates.
4 grades：Cotyledon does not stretch, yellow is serious；Hypocotyl is very thin, is bent, has infection；Root system is infected into dark brown
Color, short and small, rotten, no lateral root generates, or does not emerge seriously because root system is infected.
The calculation formula of the disease index is as follows：Disease index (DI)=∑ (series × strain number)/(highest series × total
Strain number) × 100.
Determine that the standard of eggplant resisting verticillium type is as follows according to the disease index：
If DI≤15, eggplant to be measured is disease-resistant (R) to the resistance of verticillium wilt；
If 15 DI≤30 ＜, eggplant to be measured is moderate resistance (MR) to the resistance of verticillium wilt；
If 30 DI≤50 ＜, eggplant to be measured is resistance to sick (T) to the resistance of verticillium wilt；
If 50 DI≤70 ＜, eggplant to be measured is middle sense (MS) to the resistance of verticillium wilt；
If DI ＞ 70, eggplant to be measured is susceptible (S) to the resistance of verticillium wilt.
Application of the above method in selection and breeding resisting verticillium Eggplant Varieties also belongs to protection scope of the present invention.
Beneficial effects of the present invention：
(1) compared with conventional spore suspension liquid irrigating root method, the present invention is easy to operate, saves and prepares the numerous of spore suspension
Trivial process.This method only needs vernalization to sow target variety, saves seedling management complex process.
(2) compared with conventional spore suspension liquid irrigating root method, the present invention is simply time saving and energy saving, and traditional vaccination method needs to make
It is managed again after standby spore suspension, seeding and seedling raising, seedling management, inoculation, takes typically for up to 1 month or even longer, and
The present invention only needs simply to cultivate pathogenic bacteria, vernalization sowing, and the time greatly shortens.Entire detection method can be all complete in 10 days
At.
(3) compared with conventional spore suspension liquid irrigating root method, result of the invention is more accurate, and susceptible variety is being inoculated with
Afterwards, it can fall ill after cotyledon expansion after emergence, growth deformity；And disease-resistant variety is then grown normally, no illness.
The present invention provides a kind of rapid detection methods of eggplant verticillium wilt disease resistance, and this method comprises the following steps：It is first
First the verticillium wilt pathogen being separated and identified is cultivated in PDA culture medium, after it covers with plate, the eggplant seed that will need to detect
It carries out disinfection, vernalization, and directly sows the seed after vernalization in the PDA culture medium for covering with verticillium wilt pathogen successively, on
Vermiculite after the covering high-temperature sterilizing of face, then culture medium after planting is placed in 25 DEG C of incubators and is cultivated, after cotyledon expansion, open
Begin investigation growth gesture, its Disease resistance index and resistant are determined by the growth potential of seedling.It is experimentally confirmed：The present invention's
Detection method result is accurate, and easy to operate, time saving and energy saving.
Description of the drawings
Fig. 1 is to sow aseptic germination eggplant seed picture containing bacterium culture medium and aseptic culture medium (conventional medium).A is length
The culture medium of full verticillium wilt germ；B be the germ containing verticillium wilt culture medium on sow the eggplant seed of germination；C is without germ
Conventional medium on sow the eggplant seed of germination；D is that eggplant seed after planting covers matrix.
Fig. 2 is the control performance of the sowing of normal sterile soil and aseptic culture medium sowing.A is the sowing of normal sterile soil
Control performance；B is the control performance of normal sterile culture medium sowing.
Fig. 3 is the disease-resistant sex expression of verticillium wilt of 8 kinds of eggplant cultivars.A is anti-for the verticillium wilt of Su Qi eggplant bacterium plate after planting
Characteristic of disease shows；B is the disease-resistant sex expression of verticillium wilt of black high official bacterium plate after planting；C is that the verticillium wilt of 11 bacterium plate of prestige after planting is disease-resistant
Sex expression；D is the disease-resistant sex expression of verticillium wilt of 16 bacterium plate of prestige after planting；E is anti-for the verticillium wilt of Shen eggplant No.1 bacterium plate after planting
Characteristic of disease shows；F is the disease-resistant sex expression of verticillium wilt of the black Supreme Being bacterium plate in the Northeast after planting；G is the verticillium wilt of black fairy maiden bacterium plate after planting
Disease-resistant sex expression；H is the disease-resistant sex expression of verticillium wilt of shiny black No. 1 bacterium plate after planting.
Specific implementation mode
Test method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments is respectively provided with and repeats to test three times, and results are averaged.
PDA culture medium in following embodiments is to be uniformly mixed so as to obtain 200g potatos, 20g glucose, 20g agar and 1L water
's.
Embodiment 1, a kind of rapid detection method of eggplant verticillium wilt disease resistance
1, the separation identification and culture of verticillium wilt pathogen
(1) the separation identification of verticillium wilt pathogen
The disease of the morbidity eggplant of field acquisition is good for junction and carries out alcohol disinfecting, disinfects and is placed on sterile PDA trainings
It supports and is cultivated in base, after it grows a large amount of mycelia, observation is taken pictures under the microscope first, then by mycelia tieback to healthy eggplant
Rhizome portion, after the onset of waiting for eggplant, pathogen is separately cultured from morbidity place according still further to the above method, after growing a large amount of mycelia,
It observes under the microscope, if acquired results are consistent with observation early period and " cause the separation of eggplant yellow wilting pathogen with document
With identification [J] Jiangsu's agriculture science, 2012,40 (12):The form complete one for the verticillium wilt pathogen reported in 127-129 "
It causes, then the provable pathogen is verticillium wilt pathogen.The verticillium wilt pathogen of the present invention is verticillium dahliae
Verticillium dahliae。
(2) the verticillium wilt pathogen after will be identified be seeded in PDA culture medium (specification of culture dish be diameter 80mm,
Height 10mm), be subsequently placed in constant incubator, 25 DEG C, be protected from light under the conditions of cultivate 7-10 days, so that pathogen is covered with plate and length
Gesture is vigorous uniformly, obtains bacteria-containing culture medium to be sowed (Figure 1A).
Vermiculite after soaking in order to prevent damages culture medium by pressure, convenient for seed root of hair and draws nutrient, the thickness of PDA culture medium
At least 3mm, the thickness of PDA culture medium is 3mm in the present embodiment.The present invention is when preparing PDA culture medium, into culture dish
The PDA culture medium after dissolving by heating is poured into, culture dish bottom can just not had by being subject to, and thickness is about 3mm.
2, the disinfection of eggplant seed
Eggplant seed 1-2 hour first is impregnated with sterile water, makes the pathogen on seed in the state sprouted, then with sterile
Water rinses, and rinses out a part of pathogen, the seed after being cleaned；Then the seed after cleaning is put into volume fraction is
Half a minute is impregnated in 75% ethanol water, the liquor natrii hypochloritis that mass concentration is 5% is placed into and (is purchased from Harbin City road
Outskirt Kai Yu reagents sales department) in impregnate half a minute, repeat 3-5 time, by after activation pathogen kill；Finally rushed with sterile water
It washes 2-3 times, the seed after being sterilized.
3, the sowing of eggplant seed
Vernalization is carried out under the conditions of seed after disinfection in step 2 is placed in 25-28 DEG C, obtains the seed after vernalization；And
Seed after vernalization is equably seeded in step 1 in bacteria-containing culture medium to be sowed, 50 are sowed in each culture dish
Seed obtains sowing wild Oryza species (Figure 1B).And (schemed as blank control using the PDA culture medium for not being inoculated with verticillium wilt pathogen
1C)。
4, matrix is covered
(1) pretreatment of matrix
The covering matrix of the present invention can be mixed substrate, if vermiculite and coconut palm chaff matrix are 1 according to mass ratio：1 is uniformly mixed so as to obtain
Mixed-matrix or single-matrix, such as vermiculite.
The present embodiment with vermiculite (be purchased from Zhangwu middle peasant's Sai Shi agricultural science and technologys Co., Ltd) for matrix, using autoclave by leech
Stone sterilizes 30 minutes under 121 DEG C of high temperature.And vermiculite is fully soaked using sterile water before covering (it is hand-held be atomized it is small
Watering can uniformly sprays), the matrix that obtains that treated.
(2) matrix is covered
By the seed upper layer in treated matrix cover-seeding wild Oryza species, cladding thickness is concordant with culture dish oral area to be
Can, pressable (being careful not to destroy culture medium), does not obtain the culture medium (Fig. 1 D) of covering matrix, is closed and is covered using sealed membrane
The culture medium of matrix prevents dye miscellaneous bacteria.
Above-mentioned steps 1-4 is operated in aseptic operating platform.
5, it cultivates
The culture medium for covering matrix is placed in constant incubator to cultivate, condition of culture is 25 DEG C, and culture to eggplant goes out
Sealed membrane is removed after seedling immediately, so that its normal lamina is grown culture dish opening.
Sterile eggplant seed conduct pair is conventionally sowed on sterile pure soil and sterile culture medium respectively
According to the eggplant seedling grown in control is the seedling of normal growth, to compare the eggplant seed sowed in containing bacterium culture medium
Incidence.Normal sterile soil is sowed and the control performance of aseptic culture medium sowing is as shown in Figure 2.
6, verticillium wilt Disease Resistance Identification
After the expansion of the cotyledon of eggplant seedling, emergence rate and its growing state, including cotyledon stretching, extension situation, son are investigated immediately
Leaf yellow situation, hypocotyl stretch situation, hypocotyl growth and infect situation, root growth and infect situation.According to seedling
Growth potential determines state of an illness grade, and calculates disease index.Eggplant resisting verticillium type to be measured is finally determined according to disease index.
The severity Scaling of eggplant verticillium wilt in the present invention is 0,1,2,3 and 4 grade, and specific standards at different levels are as follows：
0 grade：Cotyledon stretches；Hypocotyl is healthy and strong；Root growth is good.
1 grade：Cotyledon stretches；Hypocotyl is healthy and strong；Main root growth vigor is limited, but lateral root growth is vigorous.
2 grades：Cotyledon stretching, extension is limited, aetiolation occurs；Hypocotyl growth is very thin；Main root microbiological contamination leads to dark brown, growth
Slowly, lateral root amount is few.
3 grades：Cotyledon do not stretch, yellow；Hypocotyl is very thin；Root system main root microbiological contamination leads to dark brown, slow-growing, has a small amount of
Lateral root generates.
4 grades：Cotyledon does not stretch, yellow is serious；Hypocotyl is very thin, is bent, has infection；Root system is infected into dark brown
Color, short and small, rotten, no lateral root generates, or does not emerge seriously because root system is infected.
The calculation formula of disease index in the present invention is as follows：
Disease index (DI)=∑ (series × strain number)/(highest series × total strain number) × 100；
Eggplant resisting verticillium type to be measured is determined according to disease index：
If DI≤15, eggplant to be measured is disease-resistant (Resistance, R) to the resistance of verticillium wilt；
If 15 DI≤30 ＜, eggplant to be measured is moderate resistance (Medium Resistance, MR) to the resistance of verticillium wilt；
If 30 DI≤50 ＜, eggplant to be measured is resistance to sick (Tolerant, T) to the resistance of verticillium wilt；
If 50 DI≤70 ＜, eggplant to be measured to the resistance of verticillium wilt be middle sense (Moderate Susceptibility,
MS)；
If DI ＞ 70, eggplant to be measured is susceptible (Susceptibility, S) to the resistance of verticillium wilt.
Application of the method in detecting eggplant verticillium wilt disease resistance in embodiment 2, embodiment 1
One, material to be tested
The present embodiment selects 8 kinds of eggplant cultivars, specifying information as shown in table 1.Wherein, Su Qi eggplants are tested through forefathers and discuss
Demonstrate,prove (bibliography：Association analysis [D] the Jiangsu Yangzhou University of eggplant verticillium wilt Resistance Identification and SSR marker) it is high anti-yellowing
Disease of withering kind, remaining Eggplant Varieties are the conventional cultivation kind that market is collected.
Table 1, the title of 8 kind of eggplant cultivar and source
Two, the method for the present invention carries out verticillium wilt Disease Resistance Identification
The verticillium wilt disease resistance of the material to be tested in step 1 is identified respectively according to the method in embodiment 1.It waits for
It investigated, taken pictures after the expansion of eggplant cotyledon.
The disease-resistant sex expression of verticillium wilt of 8 kinds of eggplant cultivars is as follows：
1, the disease-resistant sex expression of verticillium wilt of Su Qi eggplants bacterium plate after planting is as follows：Leaf stretching, extension is good, no aetiolation, main root life
Long good, growth vigor is apparent, has a small amount of lateral root to generate (Fig. 3 A).
2, the disease-resistant sex expression of verticillium wilt of black high official bacterium plate after planting is as follows：Cotyledon stretching, extension is limited, there is aetiolation, main root
Growth vigor is apparent, has a small amount of lateral root to generate (Fig. 3 B).
3, the disease-resistant sex expression of verticillium wilt of 11 bacterium plate of prestige after planting is as follows：Cotyledon stretching, extension is limited, there is aetiolation, main root life
Long advantage is restricted, and generates a large amount of lateral roots (Fig. 3 C).
4, the disease-resistant sex expression of verticillium wilt of 16 bacterium plate of prestige after planting is as follows：Cotyledon stretching, extension is limited, there is aetiolation, main root
Growth vigor is restricted, and generates a large amount of lateral roots (Fig. 3 D).
5, the disease-resistant sex expression of verticillium wilt of Shen eggplant No.1 bacterium plate after planting is as follows：Cotyledon stretches critical constraints, aetiolation
Seriously, hypocotyl growth deformity, entire root system by pathogen infection cause be in dark brown, and it is short and small, have a small amount of lateral root (figure
3E)。
6, the disease-resistant sex expression of verticillium wilt of the black Supreme Being bacterium plate in the Northeast after planting is as follows：Cotyledon stretches critical constraints, aetiolation
Seriously, hypocotyl growth deformity, entire root system by pathogen infection cause be in dark brown, and it is short and small, without lateral root (Fig. 3 F).
7, the disease-resistant sex expression of verticillium wilt of black fairy maiden bacterium plate after planting is as follows：Cotyledon stretches critical constraints, and aetiolation is tight
Weight, hypocotyl growth deformity, entire root system by pathogen infection cause be in dark brown, and it is short and small, without lateral root (Fig. 3 G).
8, the disease-resistant sex expression of verticillium wilt of shiny black No. 1 bacterium plate after planting is as follows：Cotyledon stretching, extension is limited, there is aetiolation, main
Root growth advantage is restricted, and generates a large amount of lateral roots (Fig. 3 H).
Three, conventional method carries out verticillium wilt Disease Resistance Identification
Type of 8 kinds of eggplant cultivars to resistance to verticillium wilt is detected by conventional resistance to verticillium wilt detection method.Specifically
Side in step bibliography " Jiangsu association analysis [D] the Yangzhou Universitys of eggplant verticillium wilt Resistance Identification and SSR marker "
Method.
Testing result such as 2 institute of table of the verticillium wilt disease resistance detection method of the present invention and conventional resistance to verticillium wilt detection method
Show.As can be seen from the table, verticillium wilt disease resistance detection method of the invention and the testing result of common detection methods are consistent.
Illustrate the present invention verticillium wilt disease resistance detection method the result is that accurately and reliably.
The testing result of table 2, detection method and common detection methods
Claims (10)
1. a kind of detection method of eggplant verticillium wilt resistance, includes the following steps：
1) eggplant seed is seeded in the culture medium containing verticillium wilt pathogen, obtains sowing wild Oryza species；
2) matrix is covered into the seed upper layer in the sowing wild Oryza species, obtains the culture medium of covering matrix；
3) culture medium of the covering matrix is cultivated, is cultivated to the cotyledon expansion of eggplant seedling, according to the life of seedling
Growing way determines the resisting verticillium type of eggplant.
2. according to the method described in claim 1, it is characterized in that：In the step 1), the eggplant seed is gone back prior to seeding
Include the steps that disinfection and vernalization successively.
3. according to the method described in claim 2, it is characterized in that：The method of the disinfection includes the following steps：By eggplant kind
Son impregnates in sterile water, ethanol water and liquor natrii hypochloritis successively, seed after being sterilized；
Or, the method for the vernalization includes the following steps：Vernalization is carried out under the conditions of seed after the disinfection is placed in 25-28 DEG C.
4. according to the method described in claim 3, it is characterized in that：In the disinfection, time for being impregnated in the sterile water
It is 1-2 hours；The time impregnated in the ethanol water and the liquor natrii hypochloritis is 30s.
5. according to any methods of claim 1-4, it is characterised in that：The culture medium containing verticillium wilt pathogen
Preparation method includes the following steps：Verticillium wilt pathogen is seeded in the culture dish containing PDA culture medium, culture to the disease
Opportunistic pathogen covers with bacterium plate, obtains the culture medium containing verticillium wilt pathogen.
6. according to the method described in claim 5, it is characterized in that：The thickness of PDA culture medium is at least in the culture dish
3mm。
7. according to any methods of claim 1-6, it is characterised in that：
In the step 2), the matrix is vermiculite；
Or, the thickness of the matrix is concordant with the culture dish oral area；
Or, the matrix also includes the steps that sterilizing and wetting successively before covering；
Or, the condition of the sterilizing is：121 DEG C sterilize 30 minutes；
Or, the matrix is soaked using sterile water.
8. according to any methods of claim 1-7, it is characterised in that：
In the step 3), the method that the culture medium of the covering matrix is cultivated is included the following steps：It will with sealed membrane
It is cultivated after the culture medium sealing of the covering matrix, culture to eggplant removes sealed membrane after emerging, and makes its normal lamina life
It is long；
Or, the temperature of the culture is 25-28 DEG C.
9. according to any methods of claim 1-8, it is characterised in that：It is described that eggplant is determined according to the growth potential of seedling
The method of resisting verticillium type includes the following steps：Its disease index is determined according to the growth potential of seedling, further according to the state of an illness
Index determines its resisting verticillium type.
10. application of the method in selection and breeding resisting verticillium Eggplant Varieties described in claim 1-9.
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