CN107488702A - A kind of new identification sunflower method horizontal to resistance to sclerotinia sclerotiorum - Google Patents
A kind of new identification sunflower method horizontal to resistance to sclerotinia sclerotiorum Download PDFInfo
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Abstract
The invention provides a kind of new identification sunflower method horizontal to resistance to sclerotinia sclerotiorum, comprise the following steps：Step 1：Nuclear disk bacteria strain activates, step 2：It is inoculated with the preparation of matrix, step 3：Step 1 is obtained into sclerotinite inoculation in the inoculation matrix that step 2 obtains, step 4：Identify that sunflower is horizontal to resistance to sclerotinia sclerotiorum, the inoculation is cane inocalation method, prepares the matrix that sunflower cane is inoculated with as sclerotinite in step 3 in step 2.The defects of method of identification sunflower pyrenomycetes disease resistance provided by the invention is overcome in the prior art, identification is more accurate, while suitable for the identification of farmers''s strain and crosses.
Description
Technical field
The present invention establishes one kind and sclerotinite is inoculated with sunflower stalk, and identification Sunflower Varieties resist the side of (resistance to) sclerotiniose
Method, it is (resistance to) horizontal to the resistance of sclerotiniose for screening different Sunflower Varieties.
Background technology
Sunflower is one of four big oil crops in the world, China cultivated area up to 1,000,000 hm2Left and right, total yield
Measure as 192.8 ten thousand tons, be only second to rape and soybean.However, in recent years because crop rotation is difficult so that sunflower disease occurs
Area constantly expands, and Occurrence degree is also continuously increased.Sclerotium blight of sunflower is by sclerotinite (Sclerotinia
Sclerotiorum (Lib.) de Bary) caused by a kind of fungal disease, the germ is a kind of quite varied plant of host range
Thing disease fungus, the various plants of sunflower, rape, soybean, peanut, Kidney bean, carrot, arabidopsis etc. 400 can be infected.At present,
Sclerotium blight of sunflower turns into one of Major Diseases for restricting China's sunflower industry development.Sclerotium blight of sunflower is also known as white rot
Sick, rotten disk disease.This disease in sunflower main producing region per annual morbidity 50% or so, it is right up to more than 80% when serious
The yield and quality of sunflower influences very big.It is mainly shown as vertical withered type and rotten dish-type.Found withered type：Since seedling to floral disc shape
It can occur before.Seedling Stage morbidity mainly in basal part of stem, forms water soaking mode scab around stem, white flock bacterium is grown when moist
Silk, sick portion is shunk and attenuated after dry, and black sclerotium is formed in stem, and plant is in that vertical withered shape is withered.Adult plant is fallen ill also with basal part of stem
Based on, hazel moistening shape scab is showed, is gradually expanded to the stem of whole plant, it is in canescence that later stage scab is withered,
Edge is brown, skin breakage, because conducting tissue wrecks in stem, influences the transport of nutrient, blade starts from bottom to top
Gradually turn yellow, wither and come off, it is withered to eventually cause whole plant.Rotten dish-type：This illness is most heavy after thanking to the florescence, works as flower
When disk is aggrieved, occurs water soaking mode scab at its back side, holder is brown and softens.Rainy weather is met, scab expands rapidly, can wear
Pierced carving disk, front is turned to by the back side, a kind of white hypha is grown, causes floral disc to rot, seed is made when serious from maturation
Into seed benevolence rot or seed self falling
The breeding material for screening resistance to/anti-sclerotiniose is the control pathogenetic important agricultural measures of sclerotium, and indoor identification is not
It is the premise of sunflower breeding to resistance to sclerotinia sclerotiorum level with Sunflower Varieties.At present, indoor identification Sunflower Varieties are to sclerotium
The main method of sick resistance level is Isolated leaf inoculation method, and method is relatively single.And field sclerotium blight of sunflower is mostly root-rot
Type, stem rot type and disk corruption type, it is therefore desirable to establish a kind of method that new sunflower resists (resistance to) sclerotiniose.
The Chinese patent of application number 201110397710.9 discloses a kind of identification Sunflower Varieties to resistance to sclerotinia sclerotiorum water
Flat authentication method, comprises the following steps：By the sclerotinite inoculation activated, (abbreviation M is cultivated on Minum culture mediums
Base), dark culturing is accurately seeded in the both sides of sunflower blade main lobe arteries and veins then with the single mycelia agar block of acicula picking is connect,
Moisturizing culture under 20-25 DEG C of natural light is placed in, measures lesion diameter size with crossing method to identify the resistance water of sunflower
Flat, described M culture mediums contain NaOH, DL-malic acid, NH4NO3, MgSO4, agar powder.The method of the present invention is cultivated using M-
Base culture sclerotinite is inoculated with sunflower blade, can distinguish the resistance of different Sunflower Varieties, is the seed selection of sunflower new varieties
Laid the foundation with breeding for disease resistance.
In the prior art the defects of is：Sclerotiniose typically has three kinds of symptom types, i.e. brown foot rot, stem rot and disk in field
It is rotten.And it is rarely found that leaf corruption type symptom is formed on blade.Therefore, traditional blade inoculation method is caused a disease with field sunflower pyrenomycetes disease
Sign afterwards has differences, it is impossible to the pathogenic level of reaction really, the result and reality for being identified to obtain using blade inoculation
As a result can have differences, it is impossible to which identification obtains the top quality resistance to sclerotinia sclerotiorum sunflower having.In addition, blade inoculation method is by bacterium
Strain, which is seeded on blade easily to be formed, rots, and is unfavorable for the measurement of scab, causes qualification result inaccurate.
The content of the invention
The defects of in order to overcome in the prior art, the invention provides a kind of new identification sunflower to resistance to sclerotinia sclerotiorum water
Flat method, comprises the following steps：
Step 1：Nuclear disk bacteria strain activates,
Step 2：The preparation of matrix is inoculated with,
Step 3：Step 1 is obtained into sclerotinite inoculation in the inoculation matrix that step 2 obtains,
Step 4：Identify that sunflower is horizontal to resistance to sclerotinia sclerotiorum,
The inoculation is cane inocalation method, prepares what sunflower cane was inoculated with as sclerotinite in step 3 in step 2
Matrix.
Inoculation matrix has following technique effect from sunflower stalk：
1) sclerotiniose typically has three kinds of symptom types, i.e. brown foot rot in field, and stem rot and disk are rotten, and leaf is formed on blade
Rotten type symptom is rarely found.The result that inoculation cane is drawn makes qualification result more accurate closer to the Disease symptom of field sunflower
Really.
2) there is very big difference in the cell of stalk and blade composition, the inoculation matrix sick as pyrenomycetes, its moisture provided,
Nutritional ingredient and physical arrangement as matrix have differences, therefore after being inoculated with, the culture situation of bacterial strain is also different.Therefore,
Using stalk as inoculation matrix compared with being inoculated with blade, the culture in laboratory can be made to be more nearly the culture ring in field
Border, so as to get scab be more nearly field conditions, be identification result it is more accurate.
3) after stalk inoculation pathogen, the scab meeting Longitudinal Extension that germ is formed forms fusiformis scab, is easy to measure scab
Size, and be seeded on blade easily to be formed and rot, it is unfavorable for the measurement of scab.Therefore, cane inoculation is not only near to field
Disease symptom, and be also easy to observe and measure, the result of identification is more accurate.
Preferably, the sunflower cane that 6 true leaves are intercepted in step 2 is used to be inoculated with nuclear disk bacteria strain.6 true leaves
Sunflower cane is more suitable for the inoculation and culture of pyrenomycetes germ strain, and the scab after culture is more easily observable and measured.
In any of the above-described preferably, sunflower stalk is intercepted, is rinsed well, sunflower stem is wrapped up with the cotton of moistening
Bar both ends, it is placed in standby in the culture dish of sterilizing.
In any of the above-described preferably, when the interception sunflower stalk is that Sunflower Seedlings grow to 6 true leaves, from stem
Base portion intercepts 6cm sunflower cane upwards.The sunflower cane in this region is more suitable for the inoculation and culture of pyrenomycetes germ strain,
Scab after culture is more easily observable and measured.
In any of the above-described preferably, step 1 bacterial strain activation comprises the following steps：The nuclear disk bacteria strain of preservation is existed
PDA culture medium is activated, and is then beaten and is taken fresh pure culture biscuits involvng inoculation to MM culture mediums, stand-by.
In any of the above-described preferably, the PDA culture medium includes potato 200g, glucose 20g, agar powder 13g,
Distilled water is settled to 1000mL.
In any of the above-described preferably, the nuclear disk bacteria strain of preservation is 2 days in the time that PDA culture medium is activated.
In any of the above-described preferably, incubation time of the fresh bacteria cake taken on MM culture mediums is beaten from PDA as 5
My god.
In any of the above-described preferably, the MM culture mediums include NaOH 1g, DL-malic acid 3g, NH4NO3
2g, MgSO47H2O 0.1g, Bacto-agar 39g, distilled water are settled to 1000mL, and pH is adjusted to 4.7-4.8,121 DEG C of height
Flat board to be inverted on aseptic operating platform standby after pressure sterilizing 25min.Wherein, DL-malic acid are DL-malic acid, Bacto-
Agar is bacterial agar.Biology and chemical reagent used in the present invention are existing commercially available business except specified otherwise
Product.
In any of the above-described preferably, step 3 inoculation comprises the following steps：The sclerotinite that will have been grown on MM culture mediums
Colony edge card punch, which is beaten, takes bacteria cake, is inoculated on the sunflower cane got ready, and the MM culture mediums of blank compare, and are trained
Support.
In any of the above-described preferably, a diameter of 4mm of stem.
In any of the above-described preferably, condition of culture is cultivated to be placed under 22 DEG C of natural lights.
In any of the above-described preferably, incubation time 84h.
In any of the above-described preferably, the MM culture mediums configure in accordance with the following methods：NaOH 1g, DL-malic
Acid 3g, NH4NO3 2g, MgSO4·7H2O 0.1g, Bacto-agar 39g, distilled water are settled to 1000mL, pH adjust to
It is standby that flat board is inverted after 4.7-4.8,121 DEG C of autoclaving 25min on aseptic operating platform.Wherein, DL-malic acid are DL-
Malic acid, Bacto-agar are bacterial agar.Biology and chemical reagent used in the present invention are existing except specified otherwise
Commercially available commercial product.
In any of the above-described preferably, identify sunflower to sclerotium by measuring the Lesion size on cane in step 4
Sick resistance level.
There is provided a kind of new identification sunflower the method horizontal to resistance to sclerotinia sclerotiorum in a preferred embodiment of the present invention, i.e.,
Cane inocalation method, this method comprise the following steps：
Step 1：Bacterial strain is activated, and the nuclear disk bacteria strain of preservation is activated in PDA culture medium, is beaten after 2d and takes fresh bacterium
Cake is inoculated on MM culture mediums, stand-by after cultivating 5d.
Step 2：Sunflower cane prepare, when Sunflower Seedlings grow to 6 true leaves, intercepted upwards from basal part of stem 6cm to
Day certain herbaceous plants with big flowers cane, is rinsed well, is wrapped up sunflower cane both ends with the cotton of moistening, is placed in standby in the culture dish of sterilizing.
Step 3：Inoculation.The sclerotinite colony edge card punch grown on MM culture mediums is beaten to the bacterium for taking diameter 4mm
Cake, it is inoculated on the sunflower cane got ready, the MM culture mediums of blank compare, and are placed under 22 DEG C of natural lights and cultivate, are surveyed after 84h
Measure the Lesion size on cane.
Sclerotinite (Sclerotinia sclerotiorum) is the global important of a kind of damage to crops and vegetables
Phytopathogen.Sclerotinite can widely infect many unifacial leaves and dicotyledon.Preventing and treating at present to sclerotiniose is main
By chemical pesticide, but not only cost is high, pollution environment for chemical prevention, and preventive effect is also undesirable；Meanwhile the safety of food
Property is also heavily affected.Sclerotinite X-8 bacterial strains used in the present invention pick up from Baya ur, NeiMengGu city five in July, 2009
In former county sunflower experimental plot, Agricultural University of the Inner Mongol's molecule plant pathology reality is stored in after 4 DEG C through isolating and purifying and identifying
Test in room.Sclerotinite X-8 bacterial strains used in the present invention are conventional bacterial strain of the prior art, public in more academic reports
Open, herein without excessive description (such as《The research of sclerotium blight of sunflower pathogen genetic diversity and Virulence》, Wang Yu
Outstanding person, 2011 etc.).
The advantageous effects of the present invention are：A kind of new screening sunflower provided resists the method for (resistance to) sclerotiniose.
On the sunflower cane that the nuclear disk bacteria strain activated is inoculated with after MM medium cultures, determined not by cane scab length
Resistance level with Sunflower Varieties to sclerotiniose, and the Isolated leaf inoculation method that this method control is commonly used has well unanimously
Property, method that can be horizontal to resistance to sclerotinia sclerotiorum as new indoor identification sunflower.
Brief description of the drawings
Fig. 1 is the identification sunflower of the preferred embodiment of the present invention cane inocalation method and tooth in vitro horizontal to resistance to sclerotinia sclerotiorum
Piece method identifies the comparison of different Sunflower Varieties resistance levels
Fig. 2 is the identification sunflower of the preferred embodiment of the present invention cane inocalation method Lesion size horizontal to resistance to sclerotinia sclerotiorum
Statistical chart
Fig. 3 is the identification sunflower of the preferred embodiment of the present invention control group blade inoculation method disease horizontal to resistance to sclerotinia sclerotiorum
Spot size statistical chart
Embodiment：
In order to better illustrate the implementation steps of the present invention, below in conjunction with drawings and Examples, traveling one is entered to the present invention
The detailed description of step.It should be appreciated that illustrated embodiment is only used for explaining the present invention, it is not used to limit invention, it is all without departing substantially from this
The change of inventive concept is substituted within the scope of the present invention.
For trying sunflower：France 1, France 3, NX440, three eyebrows, 5009, miscellaneous No. 5 of new certain herbaceous plants with big flowers.Wherein France No. 1
Field test is high anti-sclerotiniose kind, and new miscellaneous No. 5 of certain herbaceous plants with big flowers is high sense kind.The sunflower seeds of different cultivars are seeded into and are equipped with
In the nutritive cube of high-temperature sterilization soil, it is placed in greenhouse and is germinateed, growth 4 weeks or so is stand-by.Selected by the present embodiment to day
Certain herbaceous plants with big flowers is merely to illustrate the operating method and technique effect of the present invention, but the invention is not limited in the sunflower described in the present embodiment
Bacterial strain, method provided by the present invention are applied to known Sunflower Varieties of the prior art, available for identification Local variety with
And from Local variety some Sunflower Varieties that individual plant is selected resistance level, it can also be used to identify the anti-of hybrid sunflower kind
Property it is horizontal, be not only suitable for me and cross identification of the Sunflower Lines to pyrenomycetes disease resistance level, be also applied for that there are different genetic backgrounds
Identification of the external Sunflower Lines to pyrenomycetes disease resistance level.
Bacterial strain activates：The sclerotium of the sclerotinite strain X -8 of preservation is placed in into PDA plate to be activated, beaten after 2d take it is fresh
Bacteria cake, it is inoculated on MM culture mediums, when the mycelia that sclerotial germination is formed grows to the 3/4 of culture dish, with card punch on bacterium colony side
Edge beats the mycelia block for taking diameter 4mm, stand-by.The present embodiment is carried out by taking sclerotinite strain X -8 as an example to the authentication method of the present invention
Explanation.But the invention is not limited in sclerotinite strain X -8 used in the present embodiment, in the prior art, can make sunflower
The nuclear disk bacteria strain for infecting sclerotiniose is applied to the present invention.Sclerotinite X-8 bacterial strains pick up from Inner Mongol Bayan mire in July, 2009
In your city Wuyuan County sunflower experimental plot, Agricultural University of the Inner Mongol's molecule phytopathy is stored in after 4 DEG C through isolating and purifying identification
Manage in laboratory.Sclerotinite X-8 bacterial strains are bacterial strain of the prior art, and the bacterial strain has been used for the multinomial research in this laboratory, and
Patents and article are delivered, its phenotype and hereditary feature are herein without repeating.
The preparation of sunflower excised leaf：Enamel tray (long 30cm × wide 20cm × high 5cm) disk is wiped with 75% alcohol swab
Bottom, and the blotting paper of 2 layers of sterilizing is spread in bottom, appropriate amounts of sterilized water is poured into so that blotting paper just moistens completely.Take long to 6 very
The sunflower blade of leaf, the excessive moisture of blade is blotted after rinsing well with blotting paper, then uniformly put vacuum side of blade upward
In in enamel tray.
Sunflower stalk prepares：The long sunflower plant to 6 true leaves is taken, intercepts 6cm sunflower upwards from basal part of stem
Stalk, after rinsing well, the both ends of sunflower cane are wrapped up with the cotton of moistening, are placed in standby in the culture dish of sterilizing.
Sclerotinite is inoculated with：With the single mycelia block of acicula picking is connect, the both sides of excised leaf main lobe arteries and veins, mycelia are accurately seeded in
Up；Simultaneously by inoculated by hypha block on cane, inoculation control is done with the culture block of no growth sclerotinite.Each to day
Certain herbaceous plants with big flowers kind is inoculated with 5 blades and 5 stalks, and 1 mycelia block is inoculated with each blade and stalk, not connect the agar block of bacterium work
Compareed for inoculation.Porcelain dish finally is sealed with fresh-keeping plastic foil, is placed under 22 DEG C of natural lights and cultivates.Timing observation incidence is simultaneously clapped
According to measuring the diameter (on blade) and length of scab (on cane).
Two kinds of inoculation methods can make different Sunflower Varieties blade and cane morbidities (Fig. 1 and Fig. 2).Isolated leaf inoculation
After 48h, Lesion size and length are counted after stalk inocalation method inoculation 84h.For Isolated leaf inoculation method, France No. 1 and No. 3 is in
Existing high water resistant is put down, and high sense level is presented in NX440 and miscellaneous No. 5 of new certain herbaceous plants with big flowers, and LD5009 and three eyebrow resistances are between above-mentioned two groups of resistance water
Among flat.And stalk inocalation method result also has the qualification result similar with Isolated leaf inoculation method, still, from different cultivars cane
From the point of view of upper scab length value, No. 1 and No. 3 Lesion sizes of NX440 and miscellaneous No. 5 of new certain herbaceous plants with big flowers and resistance group France differ some larger, together
When, LD5009 and three eyebrows in cane inocalation method qualification result than Isolated leaf inoculation method qualification result tend to it is disease-resistant
In kind group.It is therefore believed that stalk inoculation method, which can more accurately identify sunflower different cultivars, resists (resistance to) sclerotium
The level of disease.
With in the prior art, the authentication method of blade inoculation is compared to (identification sunflower patent CN201110397710- rooms in
The kind method horizontal to resistance to sclerotinia sclerotiorum is a patent of this laboratory in application in 2011, and its technical scheme is to utilize leaf
The authentication method of piece inoculation), the present invention has significant technique effect using stalk inoculation, further increases for sunflower
The accuracy identified pyrenomycetes disease resistance level, and this method also has significant progress in culture, measurement etc..
The advantageous effects and marked improvement of the present invention are mainly manifested in：
1) in the prior art, blade inoculation method (patent CN201110397710) mainly utilizes MM culture medium inoculateds to day
Certain herbaceous plants with big flowers blade evaluates the resistance level of sunflower, and the patent is the first patent in this laboratory, and is experimentally confirmed, and utilizes
This method predominantly detected some Local varieties and from Local variety some Sunflower Varieties that individual plant is selected resistance level.It is real
Test and show, this method can evaluate Local variety and from Local variety the different Sunflower Varieties that individual plant is selected resistance level,
But for Hybrid, this method does not have good effect.However, over time, cenospecies instead of farmers'
Position of the kind in sunflower market.Cenospecies is mainly with based on the ground import such as the U.S., Australia.These hybrid parentses
From foreign countries, genetic background is different from our Local variety, therefore finds cenospecies to sclerotium using the identification of blade inoculation method
Disease presents susceptible, or even high sense is horizontal.Authentication method provided by the present invention is not only suitable for identifying Local variety and from farmers'
The resistance level for some Sunflower Varieties that individual plant is selected in kind, it is also applied for identifying the resistance of the Sunflower Varieties of Hybrid
Level, the sunflower of the external kind of China's Local variety is different from suitable for genetic background.
2) it is difficult area that authentication method provided by the invention, which overcomes and carries out identification in the prior art in the method for blade inoculation,
The technical problem of the horizontal difference of the resistance (tolerance to diseases) of graded kind.This defect of prior art is mainly due to blade inoculation
Sequela speed is very fast, may result in all blades overnight and all rots, therefore can not measure the size of scab, evaluates
The resistance level of kind.Based on this problem, the invention provides the authentication method of cane inoculation.The groups of cells of stalk and blade
Into very big difference be present, as the inoculation matrix of pyrenomycetes disease, its moisture, nutritional ingredient and physics knot as matrix for providing
Structure has differences, therefore after inoculation, the culture situation of bacterial strain is also different.Because the fibrosis of cane are high, rotted after inoculation
Degree is low, therefore the level of the anti-sclerotiniose of different cultivars can be evaluated by the expansion rate of the scab on cane, so as to
It also avoid that blade inoculation brings the problem of being difficult to distinguish varietal resistance level difference.Therefore, it is used as inoculation base using stalk
Matter can make the culture in laboratory be more nearly the culture environment in field compared with being inoculated with blade, so as to get scab more
It is that the result of identification is more accurate close to field conditions.
3) after stalk inoculation pathogen, the scab meeting Longitudinal Extension that germ is formed forms fusiformis scab, is easy to measure scab
Size, due to being easy to observe and measuring, the result of identification is more accurate.
4) sclerotiniose typically has three kinds of symptom types, i.e. brown foot rot in field, and stem rot and disk are rotten, and leaf is formed on blade
Rotten type symptom is rarely found.Cane inoculation can more represent the cardinal symptom type (brown foot rot type) of sclerotiniose field morbidity, utilize this
The anti-sclerotiniose level of cane inoculation method evaluation Sunflower Varieties that invention provides can be more accurate compared to blade inoculation.
Explanation：The present invention according to the criteria for classifying of table one come evaluate the anti-sclerotiniose of kind it is horizontal (Wang Yujie, Gao Fengzhu,
Cao Xiong, Xu Changguo, Zhou Hongyou, Jing Lan, Zhao Jun (2010) " Sunflower Varieties resource is to identifying in resistance to sclerotinia sclerotiorum room " China
Oil crops journal (04):540-545.).
Table one：The Sunflower Varieties criteria for classifying horizontal to resistance to sclerotinia sclerotiorum
As shown in Table 1, after to be inoculated with sclerotiniose bacterial strain, through cultivating formed Lesion size as the anti-sclerotiniose of judge
Horizontal standard.When the bacterial plaque diameter X of formation is O cm, sunflower is immunological species；When the bacterial plaque diameter 0.0 of formation<X≤
During 0.5cm, sunflower is high anti-kind；When the bacterial plaque diameter 0.5 of formation<During X≤1.0, sunflower is disease tolerant variety；Work as formation
Bacterial plaque diameter X>When 1.0, sunflower is high sense kind.This evaluation method is common method of the prior art, specific inoculation
Culture and measuring method are herein without repeating.
Claims (10)
1. a kind of new identification sunflower method horizontal to resistance to sclerotinia sclerotiorum, comprises the following steps：
Step 1：Nuclear disk bacteria strain activates,
Step 2：The preparation of matrix is inoculated with,
Step 3：Step 1 is obtained into sclerotinite inoculation in the inoculation matrix that step 2 obtains,
Step 4：Identify that sunflower is horizontal to resistance to sclerotinia sclerotiorum,
Characterized in that, the inoculation is cane inocalation method, prepare sunflower cane in step 2 as nuclear disk in step 3
The matrix of bacterium inoculation.
2. the method as described in claim 1, it is characterised in that the sunflower cane that 6 true leaves are intercepted in step 2 is used to connect
Kind sclerotinite bacterial strain.
3. method as claimed in claim 2, it is characterised in that interception sunflower stalk, rinse well, with the bale of cotton of moistening
Sunflower cane both ends are wrapped up in, are placed in standby in the culture dish of sterilizing.
4. method as claimed in claim 3, it is characterised in that the interception sunflower stalk is that Sunflower Seedlings grow to 6
During true leaf, 6cm sunflower cane is intercepted upwards from basal part of stem.
5. the method as described in claim 1, it is characterised in that the activation of step 1 bacterial strain comprises the following steps：By the core of preservation
Cup fungi bacterial strain is activated in PDA culture medium, is then beaten and is taken fresh pure culture biscuits involvng inoculation to MM culture mediums, stand-by.
6. method as claimed in claim 5, it is characterised in that the PDA culture medium includes potato 200g, glucose
20g, agar powder 13g, distilled water are settled to 1000mL.
7. method as claimed in claim 6, it is characterised in that what the nuclear disk bacteria strain of preservation was activated in PDA culture medium
Time is 2 days.
8. method as claimed in claim 7, it is characterised in that training of the fresh bacteria cake taken on MM culture mediums is played from PDA
It is 5 days to support the time.
9. method as claimed in claim 8, it is characterised in that the MM culture mediums include NaOH 1g, DL-malic acid
3g, NH4NO32g, MgSO4•7H2O 0.1g, Bacto-agar 39g, distilled water are settled to 1000mL, and pH is adjusted to 4.7-
It is standby that flat board is inverted after 4.8,121 DEG C of autoclaving 25min on aseptic operating platform.
10. the method as described in claim 1, it is characterised in that step 3 inoculation comprises the following steps：Will be long on MM culture mediums
Good sclerotinite colony edge card punch, which is beaten, takes bacteria cake, is inoculated on the sunflower cane got ready, the MM culture mediums of blank are done
Control, is cultivated.
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| CN111057741A (en) * | 2019-12-31 | 2020-04-24 | 安徽农业大学 | Method for identifying resistance of kiwi fruits to canker |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433372A (en) * | 2011-12-05 | 2012-05-02 | 内蒙古农业大学 | Method for identifying resistance level of sunflower variety to sclerotiniose indoor |
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