CN110146668A - A method of measurement fungicide is to wheat stripe rust virulence - Google Patents
A method of measurement fungicide is to wheat stripe rust virulence Download PDFInfo
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Abstract
The present invention relates to a kind of measurement fungicide to the method for wheat stripe rust virulence, belongs to object protection field.The present invention is inoculated with using strip rust bacteria uredospore suspension spray, prepare fresh-keeping culture medium, tooth in vitro section chemicals treatment, detection and etc. come the inhibition situation that measures triazolone to wheat stripe rust virulence.The present invention replaces whole strain to test by the way of tooth in vitro section, is conducive to solve the problems, such as that the dosage of test sample is big and influence factor is big during the test；The present invention, which further defines the influence to spore rudiment of different antistaling agents and fresh-keeping substrate object and has studied spray, leaching medicine and drug containing mode, carries out influence after fungicide processing to the inhibiting rate of strip rust bacteria to tooth in vitro section, so that it is determined that most preferred scheme, has repeatability high and potential social and economic significance.
Description
Technical field
The invention belongs to plant protection arts, and in particular to a method of measurement fungicide is to wheat stripe rust virulence.
Background technique
For rust as the important disease endangered in Wheat Production, pathogen rest fungus is one of ten big hot research fungies.
It occurs have chronicity, fulminant, popularity and variability with harm, and effective prevention and control become long-term problem.Exist at present
It controls and subtracts usually using different measures such as agricultural methods, cultivation disease-resistant variety, chemical prevention, biological controls in actual production
Light harm.In a variety of control measures, chemical prevention is one of prevention and treatment main means of stripe rust of wheat.
Domestic and international many research units and enterprise always have stripe rust of wheat in research and innovation and screening significant anti-
Control the new type bactericide of effect.Need to carry out its sensitivity testing indoors before being put into use, and after coming into operation it is necessary
Monitoring for resistance strictly is taken, to prevent and avoid the generation and development of drug resistance, extends the service life of fungicide.And it is novel
It is the basis for instructing field medication to the sensitivity base-line of fungi that fungicide is measured in medicament chamber, and can reflect fungicide and exist
The fitness and development trend of germ after use.It is this for wheat stripe rust by air-flow propagate obligatory parasitism fungi and
Speech, studies reliable and stable, operates convenient and repeated high toxicity test method, is Screening of Fungicide and drug resistance research and controls
The premise of reason.
For obligate parasite, it is different from non-obligate parasite, mycelia cannot be detached from host's growth, spore germination
Mycelia in the space between cells of host plant mainly by extending afterwards, and pathogen is difficult to save under ex vivo.Therefore, it passes
The mycelial growth rate method and spore germination method of system are difficult to effectively measure medicament to the practical virulence effect of germ.At present for
The method of the sensitivity base-line measurement of Wheat rust fungus has dressing seed method, Medicament soaked seed method, living body pot-spraying method etc., but each
The germ measured between kind of method is affected by other factors that gap is larger, and Medicament soaked seed and seed dressing are general to the sensibility of fungicide
Start to be inoculated with when wheat seeding is fully deployed 1 leaf, reaches measurement standard to control group production spore and just start to calculate drug effect, is i.e. medicament is made
It has been up to after 15d with the time and has been measured again, this is more demanding for the lasting period of medicament, and pharmacy effect is by other factors
Being affected including soil and moisture.With living body potting by spraying, investigation as a result mostly uses estimation lesion area, conversion
At disease index, in the control efficiency for calculating each medicament.Impacted factor is more during the test for these methods, including medicine
Agent dosage form and dosage, plant growth situation etc., repeatability is not high, and the measurement method of data is larger by human factor, and work
Work is measured and vegetable material needs to reach a certain amount to adjust the reasonability of data.
Measurement in a kind of wheat powdery mildew room is disclosed in world fungicide resistance Action Committee (FRAC) at present to sterilize
The method of agent medicament first carries out reagent spray processing to potting wheat seedling, and then clip drug containing leaf section is placed in fresh-keeping culture medium
Spore sedimentation inoculation is carried out, number sorus number carries out the calculating of preventive effect after culture；And measurement cucumber downy mildew bacterium
The method of power is medicament suspension leaf dish, then the method being accurately inoculated with.The above method shows the drug effect for obligate parasite
Virulence measurement method has feature development easy to operate, repeatability is high and workload is small.But it is directed to Wheat rust fungus at present
To the measurement method of fungicide, FRAC announce be or living body pot-culture method.
It can thus be appreciated that there is an urgent need to establish the method for a kind of accurate and effective monitoring fungicidal activity, to determine and compare
Different medicines are to the fungistatic effect and virulence of wheat rust, and the monitoring of the screening and field resistance bacterial strain for new type bactericide provides
Theory- method-technology is supported.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of measurement fungicide to the method for wheat stripe rust virulence.
In order to achieve the above objectives, the invention provides the following technical scheme:
A method of measurement fungicide is to wheat stripe rust virulence, and described method includes following steps:
(1) inoculated and cultured: the wheat stripe rust spore suspension of 5~10 μ g/mL is inoculated on wheat seedling, in dark
It takes out, cultivates in the greenhouse spare after scab to occurring after moisturizing processing；
(2) it prepares fresh-keeping culture medium: antistaling agent and fungicide being added in water agar and are uniformly mixed, culture dish is poured into
In, then cover the fresh-keeping culture medium that drug containing is prepared in filter paper；
(3) tooth in vitro section chemicals treatment: there is the wheat seedling of scab in (1) in selecting step, by the identical difference of growing way
The interlude with obvious scab in basin is as tooth in vitro section, the filter being placed in the fresh-keeping culture medium of drug containing described in step (2)
On paper, after the both ends of the tooth in vitro section are fixed, then it is placed in insulating box and cultivates；
(4) it detects: measuring sorus accounting using photoshop, calculate control efficiency and EC50。
Preferably, the preparation method of wheat stripe rust spore suspension described in step (1) are as follows: by wheat stripe rust conidia powder
Be added in 0.1% polysorbas20, continue concussion it is fully dispersed to conidia powder, spore suspension can be prepared.
Preferably, seedling described in step (1) is the wheat seedling of 1 leaf phase of 1 heart.
Preferably, the time of the processing of moisturizing described in step (1) is that for 24 hours, the condition cultivated in the greenhouse is 17~20
DEG C, illumination 16h/d.
Preferably, the tool used when being inoculated with described in step (1) is airbrush, pressure setting of the airbrush in inoculation
For 0~0.4bar, when inoculation, is inoculated with according to the amount of every basin wheat seedling 2mL suspension.
Preferably, antistaling agent described in step (2) includes appointing in gibberellin, 6- benzyl aminoadenine or benzimidazole
It anticipates one kind.
Preferably, the mass volume ratio of the gibberellin and water agar is 10~30:1, the 6- benzyl aminoadenine with
The mass volume ratio of water agar is 25~100:1, and the mass volume ratio of the benzimidazole and water agar is 50~100:1, institute
The unit for stating quality volume is μ g/mL.
Preferably, fungicide described in step (3) is triazolone, the ratio between the triazolone and water agar for 0.03~
21.87:1 μ g:mL.
Preferably, described method includes following steps:
(1) inoculated and cultured: wheat stripe rust conidia powder being added in 0.1% polysorbas20, clear water is added after concussion, after
Continuous concussion is fully dispersed to conidia powder, and spore suspension, the mass body of conidia powder described in the spore suspension can be prepared
Product concentration is 5~10 μ g/mL；
(2) it prepares fresh-keeping culture medium: 6- benzyl aminoadenine and triazolone being added in 0.5% water agar and mixed
It is even, pour into culture dish, the mass volume ratio of the 6- benzyl aminoadenine, triazolone and water agar be 75:0.03~
G: μ g:mL of 21.87:1, μ；
(3) circular filter paper of 9cm diametrically tooth in vitro section chemicals treatment: is cut into 2.5cm, 4.0cm, 2.5cm first
Three sections, the filter paper item of intermediate 4cm is spread in the culture dish being added in step (2)；Next, which takes in step (1), there is the wheat of scab
The wheat of the different basins of the identical sustained height of growing way is chosen in seedling potting, the conduct with obvious scab of 5cm among clip
Leaf section both ends are fixed with remaining 2.5cm filter paper on the filter paper of 4cm and are allowed to be tightly attached to culture by tooth in vitro section, parallel laid
Primary surface makes containing 5 leaf sections from different basins in each culture dish, then culture dish is placed in light in 12 DEG C of constant incubators
Test group is formed according to culture 18h；
(4) control group: as a control group with not adding of germicide, other conditions and the identical culture dish of test group；
(5) it detects: carrying out scab accounting pixel analysis with Photoshop software, first record total pixel of 5 leaf sections, then
Record total pixel of sorus area in 5 leaf sections under 50,60,70 3 tolerances；Spore in 5 leaf sections is calculated with Excel
The average value and sorus area accounting of total pixel of sub- heap area.
The beneficial effects of the present invention are:
1, spore is propagated by air-flow to achieve the purpose that diffusion due to wheat stripe rust, so needing when purifying and expanding numerous
It is isolated culture, prevents and treats cross contamination, to be collected into the strain of purifying, as obligate parasite, the summer of strip rust bacteria in nature
The holding time of spore at normal temperature is shorter, needs store method appropriate to extend the holding activity time of spore.Fungicide
What is mostly used to the toxicity test of stripe rust of wheat is whole plant inoculation pathogen separation object, and this mode dosage is big, and by it
His factor influences big.So the present invention replaces whole strain to test by the way of tooth in vitro section, it is big to can solve above-mentioned dosage
And the problem that influence factor is big.
2, since wheat leaf blade is tiny, the fresh-keeping work of moisturizing is that bioassay first has to solve the problems, such as.Protect green culture
The ingredient (sark and green-preserving agent) of base protects the influence and tooth in vitro of green time and green-preserving agent to the germination rate of inoculation bacterium spore
The leaf age of piece itself, the factors such as length are all the objects of research.Amid all these factors, the present invention has studied antistaling agent and fresh-keeping
Influence of the substrate object to spore rudiment, it is determined that most preferred scheme, i.e., using the 6-BA of 75 μ g/mL as green-preserving agent, in conjunction with 0.5%
Water agar and 1 layer of filter paper composition be fresh-keeping culture medium of the sark as wheat leaf section.
3, require the influence factor of various aspects in plant and germ interaction system that can reach optimal combination shape when being inoculated with
State will also be conducive to plant living body and keep best vigor, and have repeatability, in this way even if germ plays maximum pathogenicity
Just it is conducive to the accuracy of subsequent pharmacy test.Studies have shown that the common inoculation method of rest fungus includes artificial friction, brush leaf method (is used
Writing brush or cotton swab smear conidia powder or spore suspension), spray-on process, powder injection process, and come by mechanical inoculation cylinder or sedimentation tower
Carry out quantitative inoculation.The present invention sets out from convenience, efficient angle, establishes the spore suspension effective concentration of inoculation in 5-10 μ g/
Between mL, and by HD-130 airbrush come carry out even spraying inoculation in the way of.
4, medicament preferably acts on strip rust bacteria and exercising result is relatively stable in order to allow, repeatability operation.This
Invention equally has studied spray, leaching medicine and drug containing mode and carries out inhibition to strip rust bacteria after the fungicide processing of tooth in vitro section
Rate, select drug containing mode it is preferred that, fungicide can be made preferably to act on strip rust bacteria in the tooth in vitro section of wheat,
Germ to the sensibility of medicament close to living body wheat medicament spraying treatment and result it is relatively stable, while relative to traditional work
Body potting is by spraying and for dressing seed method, and pharmacy effect is more accurate in the effect of strip rust bacteria, and vegetable material dosage is few.
5, the present invention calculates in such a way that uredium area accounting of the Photoshop to leaf section carries out precise measurement
The bacteriostasis rate of fungicide is estimated the mode of disease severity relative to currently used staging, is influenced by human factor
Smaller, the result for obtaining its test is more acurrate.
Other advantages, target and feature of the invention will be illustrated in the following description to a certain extent, and
And to a certain extent, based on will be apparent to those skilled in the art to investigating hereafter, Huo Zheke
To be instructed from the practice of the present invention.Target of the invention and other advantages can be realized by following specification and
It obtains.
Detailed description of the invention
To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention is made below in conjunction with attached drawing excellent
The detailed description of choosing, in which:
Fig. 1 is fresh keeping time of the wheat 1cm tooth in vitro section in different antistaling agents under various concentration, and wherein GA is indicated red mould
Element, BEN indicate that benzimidazole, 6-BA indicate that 6- benzyl adenine, the numerical value in abscissa bracket indicate each antistaling agent
Concentration (μ g/mL)；
Fig. 2 is the different green sarks of guarantor to the green effect of guarantor of Vitro In Wheat leaf section, the green liner of guarantor that wherein A, B, C, D are indicated
Object is respectively 0.5% water agar and composition, 0.5% water agar, 1% water agar and the 1 layer of filter paper of the formation of 1 layer of filter paper
The composition of formation, 2 layers of filter paper, and E indicates the blade (control group) of potting wheat；
Fig. 3 is the morbidity feelings of wheat leaf blade after spray inoculation 0 (CK), 0.5,1,5, the uredospore suspension 15d of 10mg/ml
Condition；
Fig. 4 is 1 μ g/ml and 5 μ g/ml triazolones pass through spray, leaching three kinds of processing modes of medicine and drug containing to wheat item respectively
The control efficiency of rest fungus, wherein A is the effect of chemical control with watering can spray liquor on the potting wheat being inoculated with after 9d, and B is
To be inoculated with the wheat leaf blade after 9d it is in vitro after be immersed in the effect of chemical control for being put into and protecting in green culture medium after 2h in medical fluid, C is
Effect of chemical control wheat leaf section after inoculation 9d being put into after in vitro in the green culture medium of guarantor of drug containing；
Fig. 5 is chemicals treatment schematic diagram of the plate drug containing method to tooth in vitro section；
Fig. 6 is that Photoshop handles Vitro In Wheat leaf section uredium accounting schematic diagram；
Fig. 7 be measured using plate drug containing method four wheat stripe rust monospore heap isolated strains (Pst-20, Pst-31,
Pst-26, Pst-21) various concentration triazolone processing under growing state；
Fig. 8 is EC of 81 wheat stripe rust monospore heap isolated strains to triazolone50Standard Q-Q figure；
Fig. 9 is sensitivity base-line of the 81 monospore heap isolated strains of strip rust bacteria to triazolone；
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.It should be noted that diagram provided in following embodiment is only to show
Meaning mode illustrates basic conception of the invention, and in the absence of conflict, the feature in following embodiment and embodiment can phase
Mutually combination.
The various raw material sources used in the present invention are as follows:
Test plant material: wheat breed is inscription virtuous 169, is wheat stripe rust height sense kind.Sowing is until the first leaf is complete
Test process is used for when full expansion.
Strains tested: strain is the acquisition of the Hechuan District of Chongqing in 2018 field (north latitude 30.10'24 ", east longitude 106.25'37 ")
Mixed bacteria.
For agent of having a try:
Gibberellin (GA): weighing 0.1g, after the dissolution of 500 μ L alcohol is added, adds the dilution of 49.5mL distilled water, is stored in
In brown bottle, it is placed in 4 DEG C of refrigerators and saves；
6- benzyl aminoadenine (6-BA): it weighs 1g 6-BA and is placed in small beaker；It is added 0.5 a small amount of or 1mol/L's
Hydrochloric acid is allowed to be completely dissolved；100mL, 4 DEG C of vial preservations are being diluted and are being settled to distilled water；
Benzimidazole (BEN): it weighs 1g benzimidazole and is placed in small beaker；The hydrochloric acid of 0.5 a small amount of or 1mol/L is added,
It is allowed to be completely dissolved；100mL, 4 DEG C of vial preservations are being diluted and are being settled to distilled water；
95% triazolone raw medicine (is purchased from Hubei Kang Baotai company), weighs 0.5g, using 5mL dimethyl sulfoxide as solvent, matches
The mother liquor of 1 × 104 μ g/mL is made, is placed in 4 DEG C of refrigerator and saves backup.
For trying culture medium: the preparation of 0.5% water agar (WA) culture medium: taking 300mL conical flask, weigh the agar powder of 1g,
The sterile water of 200mL is added, sealing, which is placed in high-pressure sterilizing pot, to sterilize, spare after cooling.
Embodiment 1 prepares test cultures ware
1, the separation of wheat stripe rust bacterial strain and expansion are numerous
(1) wheat planting: firstly, choosing full consistent virtuous 169 wheat seed of inscription, 1 is impregnated with 500mL beaker dress clear water
Night after the abundant water swelling of seed, pours into the culture dish that diameter is 25cm, and clear dry surface moisture covers wet gauze, dress
Enter in black plastic bag, is placed in vernalization in 25 DEG C of insulating boxs；Secondly, adding after mixing peat soil and vermiculite after seed shows money or valuables one carries unintentionally
Water-wet profit, is layered in the small plastic flowerpot of 7 side of black, and the wheat seeds that show money or valuables one carries unintentionally for choosing health are sprinkled into, and 1 basin is put into 15~20 seeds,
One layer of compost is covered again, is sprinkled profoundly water, (16h/8h illumination/dark) is placed in greenhouse, grows to 1 heart, 1 Ye Shibei to 10d
With.
(2) acquisition of wheat stripe rust: acquiring sick leaf first, originates the phase in stripe rust of wheat and adopts from different wheat ecology areas
Sample, acquires fresh stripe rust disease leaf, selects the biggish sick leaf of wheat leaf surface sorus spacing, the leaf section of clip monospore heap,
It is wrapped with blotting paper, is entirely clipped in book, acquisition information has been marked at footer, taken back and be put in preservation in 4 DEG C of driers；Its
Uredospore is acquired, by the teat glass that the leaf section insertion of monospore heap is dry, tapping tube wall shakes off the conidia powder on blade, uses
Sealed membrane sealing, it is labelled, it indicates locality and the time, takes back to be transferred to centrifuge tube and vacuumize and be stored in -80 DEG C of refrigerators.
(3) strip rust bacteria single spore separation (just numerous): carrying out moisturizing processing for the sick leaf taken back, and the sorus for choosing acquisition relatively divides
Scattered leaf section is layered in the culture dish with wet filter paper overnight after rinsing surface with clear water, puts 3 to 5 in each culture dish
Blade prevents cross-infection, spare after the new spore of output.
Choose the healthy wheat seedling for sowing 1 plant of 1 cave of the 10d in seedlings nursing plate in advance.It is first spraying to workbench before inoculation, it uses
To settle the puccinia striiformis spore in air, then carried out disinfection to transfer needle and hand with 75% alcohol.It chooses at moisturizing
Single spore heap on the sick leaf of reason dips the spore on single spore heap with the inoculation needle tip of sterilizing, gently moves needle point
On to wheat leaf blade to be seeded, it is seeded in middle part of blade as far as possible, spraying a small amount of clear water after being inoculated with, with transparent suction cover
Firmly, it is isolated.It takes another 1 transfer needle to dip another 1 sorus again, is so repeatedly performed the separation inoculation of monospore heap.1 spore
Sub- heap is inoculated with 1, and 1 sick leaf is inoculated with 3 plants of wheats.Incidence is observed after 12~15d of hot-house culture.
(3) strip rust bacteria bacterial strain expands numerous: firstly, cutting blade after the blade of monospore heap separation produces spore, being put into inoculation ware
In, a small amount of clear water stirring is added, falls the spore on blade, spore suspension is made after filtering；Secondly, choosing health and growing way one
The potting 10d wheat seeding of cause, finger dips clear water and rubs blade 2-3 times, and to remove surface wax coat, airbrush is added in spore suspension
In, spray inoculation is on processed wheat seedling, then is sprayed a small amount of clear water with watering can and moistens blade, label is plugged, then with modeling
Material sleeve covers isolation, and dark processing is taken out afterwards (after having handled 1 bacterial strain, 75% alcohol is added into airbrush for 24 hours in the greenhouse
Spraying 10s, adds clear water and is sprayed 10s, after station settles 120s, carries out the processing of lower 1 bacterial strain)；Finally, every 5d
Lobus cardiacus is cut off, a small amount of clear water is poured, dry centrifuge tube collects spore when producing spore, numbers respectively spare in cryo-conservation.
(5) preservation of strip rust bacteria bacterial strain: it is dry in 4 DEG C of refrigerators that uredospore will fill the Restored in test tube of spore during collecting
In dry device.Uredospore is fitted into centrifuge tube to vacuumize after the completion of collection and is stored in -80 DEG C of refrigerators.
2, the screening of antistaling agent
It is green in order to protect wheat leaf blade under ex vivo, to meet the onset condition of wheat stripe rust, therefore use water outstanding
Floating method carries out the screening of the fresh-keeping single dose of Vitro In Wheat leaf section: after carrying out station disinfection with 75% alcohol, Xiang Shengyou goes out
Different amounts of fresh-keeping single dose is added in the conical flask of bacterium water, then configured fresh-keeping aqueous solution is poured into the culture dish of 9cm and is made
At water pond.The wheat leaf section for choosing consistent 1 leaf, 1 heart of healthy growing way, is cut into the leaf section of 1cm or so, every ware is put on station
10 leaf sections are set, middle period Duan Jun is that face-up, sealing is placed in wheat greenhouse.As follows, gibberellin is arranged in fresh-keeping single dose concentration
It (GA) is 10,20,30 μ g/mL；6- benzyl aminoadenine (6-BA) is 25,50,75,100 μ g/mL；Benzimidazole (BEN) is
50,65,75,100 μ g/mL, using water as control group, each processing is repeated 3 times, and observes leaf section yellow situation daily, and record is opened
As a result the time of the time of beginning yellow and complete yellow screen Vitro In Wheat leaf section by the method for aqueous suspension as shown in Figure 1:
Antistaling agent, three kinds of antistaling agents have better tooth in vitro section to protect green effect for water, wherein the 6- benzyl of 75 μ g/mL
Time longest required for safety guarantor's green time of adenine and complete yellow, respectively 12 days and 19.33 days；Benzimidazole
It protects green effect to take second place, the guarantor of the benzimidazole of 100 μ g/mL can reach 16 days the green time；The green effect of the guarantor of gibberellin is worst, and 30
The gibberellin of μ g/mL can only protect green 14.67d.Since wheat stripe rust can all be grown wheat leaf section before complete yellow, and
Significant result analysis shows that, all there is significant difference in the 6- benzyl adenine of 75 μ g/mL and the green-preserving agent of other concentration,
It therefore is preferred green-preserving agent in the present invention by the 6- benzyl adenine of 75 μ g/mL.
3, the screening of fresh-keeping sark
Using the 6-BA of 75 μ g/mL as antistaling agent, tested respectively with the combination of 0.5% water agar and the formation of 1 layer of filter paper
The composition (C) and 2 layers of filter paper (D) that object (A), 0.5% water agar (B), 1% water agar and 1 layer of filter paper are formed are fresh-keeping
The fresh-keeping situation of substrate object, as a control group by the blade (E) of potting wheat, test result is as shown in Figure 2: each component has one
The green effect of fixed guarantor, the composition formed with 0.5% water agar and 1 layer of filter paper is best for the fresh-keeping effect of fresh-keeping substrate object,
Just start yellow, complete yellow after in vitro 19d after the in vitro 12d of leaf section；0.5% water agar is the green effect of guarantor of fresh-keeping substrate object
It is worst, with regard to complete yellow after the in vitro 15d of leaf section.It can be seen from the above result that the composition that 0.5% water agar and 1 layer of filter paper are formed
There are significant differences for the green effect of guarantor of the green effect of guarantor and other several fresh-keeping substrate objects when for fresh-keeping substrate object, it is possible to
Optimal case using 0.5% water agar and the composition of 1 layer of filter paper formation as the fresh-keeping substrate object of Vitro In Wheat leaf section.
4, influence of the green culture medium to uredospores germination is protected
After choosing the preferred combination of antistaling agent and fresh-keeping sark, then determines and protect green medium component to wheat stripe rust
The influence of the sprouting of uredospore: taking 3 clean concave slides, is separately added into the fresh-keeping liner of 250 μ L with liquid-transfering gun in a groove
Object, including sterile water, 0.5% water agar, and the 0.5% water agar of 75 μ g/mL 6-BA is added；Take 10mL clean
Centrifuge tube weighs the uredospore powder of proper amount of fresh, and 3 times of talcum powder is added, and is sealed after mixing with two layers of gauze, is shaken off from top
It is put into moisturizing in porcelain dish to slide, after number, is placed in dark culturing in 12 DEG C of incubator；Again after microscopy culture 3h and 6h
Concave slide: slide being placed under microscope (20 × 10) and is observed, and each concave slide observes 10 visuals field, record uredospore sum and
It sprouts spore count (germ tube is more than that spore minor axis 1/2 is to sprout) and calculates spore germination rate (%), test is repeated four times.
Wherein, spore germination rate (%)=sprouting spore count/uredospore sum × 100%
It is control with sterile water, influence of the green culture medium to uredospores germination is protected in measurement, and the results are shown in Table 1, shows to protect
The 6-BA of 75 μ g/mL of 0.5% water agar of ingredient and green-preserving agent in green culture medium is on uredospores germination without influence: under 3 kinds of processing
Germination rate of the spore after 6h is attained by 80% or more, and three groups of data, and there was no significant difference.
Influence of 1 green-preserving agent of table to wheat stripe rust uredospores germination
Note: in table after data different letters indicate to show using Duncan otherness between duncan's new multiple range method examines numerical value (P >
0.05)
5, it is inoculated with the uniformity and inoculum concentration determines
Firstly, first 2min is stood, to settle in air to the alcohol of station spraying 75%, then spraying a small amount of clear water
Spore；Secondly, weighing the conidia powder of 5mg, 0.02% polysorbas20 of 10mL is added, 5min is vibrated on turbula shaker, makes
It is 0.5mg/mL spore suspension at concentration, same method and reagent configuration concentration are 1.0mg/mL, 5mg/mL and 10mg/mL
Spore suspension；Then, by the spore suspension made be added bore be 0.5mm Spray pen for painting in, adjust Spray pen for painting pressure be 0~
Spray inoculation is carried out after 4bar, 5mL bacterium solution is inoculated with 3 basin seedling, to be inoculated with 0.02% polysorbas20 (CK) as control group；It is last dark
Moisturizing processing is placed on hot-house culture for 24 hours, records incubation period and the incidence of 9d, 12d, 15d and 18d.By 3 when being wherein inoculated with
Piece coverslip is placed in culture ware lid and is placed in by inoculation seedling, spraying together.After the completion of inoculation, coverslip is placed in microscope (10
× 10) it is observed under, each glass slide observes 5 visuals field.
In formula:
I=F × D × 100
D --- sick leaf is averaged severity；I --- each serious angle value；
li--- the corresponding sick number of sheets of each serious angle value, unit is piece；
L --- investigation total number of sheets, unit is piece；I --- disease index；
F --- sick leaf rate.
As the result is shown: the different spore suspension of 4 kinds of concentration is remembered after carrying out spray inoculation with optical microscopy (10 × 20)
The spore count in 5 visuals field on each slide has been recorded, has used the duncan's new multiple range method of SPSS software to analyze data, each group after calculating average
As a result as shown in table 2 below: simultaneously there was no significant difference between 3 coverslips in each group of data group, shows bacteria suspension when spray inoculation
It is evenly distributed；The incidence of wheat leaf blade is such as after spray inoculation 0 (CK), 0.5,1,5, the uredospore suspension 15d of 10mg/ml
Shown in table 3 and Fig. 3: concentration is that the spore suspension of 1~10 μ g/mL can be effectively inoculated with, and the concentration of spore suspension with
The disease incubation period is related, i.e. the concentration of uredospore is higher, and the incubation period of disease is shorter, and with the spore of 5 μ g/mL and 10 μ g/mL
There was no significant difference for disease index of the wheat of suspension inoculation after 15d, therefore can choose 5~10 μ g/mL as effectively inoculation
The concentration of effective spore suspension of wheat stripe rust.
Every visual field of airbrush spray inoculation is averaged spore count under 2 optical microscopy of table (10 × 20)
Note: in table after data different letters indicate to show using Duncan otherness between duncan's new multiple range method examines numerical value (P >
0.05)
Disease incubation period and disease index under 3 spray inoculation various concentration spore suspension of table
Note: in table after data different letters indicate to show using Duncan otherness between duncan's new multiple range method examines numerical value (P >
0.05)
6, tooth in vitro section chemicals treatment
In order to enable fungicide to play one's part to the full, and effect stability can be made and repeated, compare wheat tooth in vitro section
Spray soaks 3 kinds of modes of medicine and plate drug containing to the function and effect of wheat stripe rust, wherein with the triazole of 1 μ g/mL and 5 μ g/mL
Ketone is as fungicide: firstly, the uniform leaf section of scab situation is generated after choosing inoculation, use after tooth in vitro section is put into freshness preserving plate
Small watering can carries out spray processing (being uniformly covered with leaf section and non-condensing for until water droplet using medicine drop) afterwards as spray group, by inoculation
Leaf section cut immerse medical fluid 2h after use filter paper to wipe dry surface drop after be put into freshness preserving plate as soak medicine group, directly medicament is added
Drug containing tablet is made in fresh-keeping culture medium, the leaf section for producing spot is put into drug containing tablet as plate drug containing group, 3 kinds of processing modes
It was carried out with 1 day, and selection is consistent；It is cultivated secondly, the plate handled well is put into incubator, with what is handled without fungicide
Tooth in vitro section is as a control group (i.e. the concentration of triazolone is 0 μ g/mL)；It is accounted for finally, calculating sorus area using Phtotshop
Than.As a result as shown in Figure 4: the Vitro In Wheat leaf section with strip rust bacteria is passed through the spray of fungicide, is soaked at 3 kinds of modes of medicine and drug containing
After reason can control efficiency be generated to strip rust bacteria, wherein when either the triazolone of 1 μ g/mL or 5 μ g/mL are as fungicide,
There was no significant difference with pot-culture method for the control efficiency that the processing mode of plate drug containing obtains, therefore at the fungicide of tooth in vitro section
Plate drug containing is selected to can replace the control efficiency that pot-culture method obtains it to strip rust bacteria in reason mode.
The application test of 2 tooth in vitro section plate drug containing of embodiment
In conjunction with the preferred result of above-described embodiment 1, wheat stripe rust monospore heap isolated strains are randomly selected, using plate
Drug containing method carries out triazolone to the toxicity test of wheat stripe rust, and the specific method is as follows:
(1) it prepares spore suspension: the monospore heap isolated strains for being stored in -80 DEG C being taken out, are placed in 42 DEG C of water-baths
Activation 5min obtains conidia powder, and appropriate conidia powder is poured into 10mL centrifuge tube, 0.1% polysorbas20 of 1mL is added, is being vortexed
9mL clear water is added after shaking 1min on oscillator, shakes 4min or so again, until conidia powder is fully dispersed, it is outstanding that spore is made
Supernatant liquid.
(2) it is inoculated with: first to the alcohol of station spraying 75%, then spraying a small amount of clear water, 2min is stood, to settle air
In spore；The spore suspension made is added in the Spray pen for painting that bore is 0.3mm, is carried out under the pressure of 0~4bar
Wheat seedling inoculation, each 5 basin potting seedling of bacterial suspension inoculation guarantee that blade two sides is paved with droplet and does not drip (its when inoculation
In it is every be inoculated with 1 bacterial strain after, clean airbrush with clear water, the alcohol for adding appropriate 75% is sprayed 10s, is finally rushed again with clear water
It washes, can just carry out the inoculation of lower 1 bacterial strain)；Inoculated seedling potting is placed in a greenhouse dark moisturizing processing to take afterwards for 24 hours
Out, kept apart with plastic sleeve, be placed in a greenhouse culture, during which remove lobus cardiacus, to guarantee the 1st leaf nutrition supply, until 8d
There is scab in left and right.
(3) in vitro chemicals treatment: wheat seedling when obvious scab occur but not starting to produce spore is carried out in vitro medicament
The 6-BA and triazolone of corresponding amount are added in 0.5% water agar (WA) culture medium for being cooled to non-scald on hand, is configured to contain for reason
The training of the triazolone of 75 μ g/mL 6-BA and various concentration (0.03,0.09,0.27,0.81,2.43,7.29,21.87 μ g/mL)
Base is supported, the culture medium for then taking 20mL to prepare pours into the culture dish of 9cm；The circular filter paper of 9cm is diametrically cut into 2.5,
The filter paper item of intermediate 4cm is layered on culture medium by three sections of 4.0,2.5cm, chooses the wheat leaf blade of the different basins of sustained height,
For the intermediate leaf section with obvious scab of clip 5cm as tooth in vitro section, parallel laid, will with remaining filter paper on 4cm filter paper
Leaf section both ends are fixed and are allowed to be tightly attached to media surface, it is desirable that contain 5 leaf sections from different basins in each culture dish；Place
After having managed, culture dish is placed in 12 DEG C of constant incubators (illumination 18h) and is cultivated, is so that the not pastille culture medium of triazolone is not added
Control group (as shown in Figure 5).
(5) it detects: being infected when the scab of control group and carry out data statistics when area reaches 80% or more, use Photoshop
Software carries out scab accounting pixel analysis, first records total pixel of 5 leaf sections, re-records 5 under 50,60,70 three tolerances
Total pixel of sorus area in leaf section；With Excel calculate in 5 leaf sections the average value of total pixel of sorus area and
Sorus area accounting (as shown in Figure 6)；Logarithm with drug concentration is X, probability value corresponding to the inhibiting rate of strain growth
For Y, the virulence regression equation Y=aX+b of bacterial strain is calculated；As Y=5, the EC of each bacterial strain is calculated50, wherein inhibiting rate
(%)=(control group sorus area accounting-processing group sorus area accounting)/control group sorus area accounting ×
100%；With SPSS software to EC50Analysis and test of normality is described, by the EC of bacterial strain50As X-axis, distribution frequency
For Y value, a frequency histogram distribution map is obtained, Trendline is sensitivity base-line, and obtained sensitivity base-line is carried out just
State property Shapiro-Wilk (W inspection), and further generate Q-Q figure.
Test result measures 81 monospore heap isolated strains (as shown in Figure 7) to the EC of triazolone altogether in aforementioned manners50,
The EC that will be obtained50It carries out normality Shapiro-Wilk (W inspection), as the result is shown to the frequency of triazolone difference sensitive strains
Rate distribution is in approximate normal distribution (W=0.204), and further generates Q-Q figure, specific as shown in figure 8, each EC as the result is shown50
Value surrounds lineal layout, is in normal distribution.
In conclusion EC of 81 bacterial strains measured to triazolone50Variation range be 0.0015~0.3117 μ g/mL,
Average value is 0.1453 ± 0.0081 μ g/mL, frequency distribution (group away from being normal state unimodal curve for 0.02 μ g/mL) (shown in Fig. 9),
So can be by measured EC50Average value as stripe rust of wheat to the sensitivity base-line of triazolone, to obtain measurement fungicide
To the independent method of wheat stripe rust.It, can be in the feelings that antistaling agent and fresh-keeping substrate object is added due to passing through verifying of the invention
Under condition, wheat potting is replaced using three-dimensional leaf section, therefore method of the invention can also be directly used in indoor measurement fungicide to small
The virulence effect of wheat strip rust bacteria.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of the technical program, should all be covered in the present invention
Scope of the claims in.
Claims (9)
1. a kind of measurement fungicide is to the method for wheat stripe rust virulence, which is characterized in that described method includes following steps:
(1) inoculated and cultured: the wheat stripe rust spore suspension of 5~10 μ g/mL is inoculated on wheat seedling, the moisturizing in dark
It takes out, cultivates in the greenhouse spare after scab to occurring after processing；
(2) it prepares fresh-keeping culture medium: antistaling agent and fungicide being added in water agar and are uniformly mixed, is poured into culture dish, then
The fresh-keeping culture medium of drug containing is prepared in covering filter paper；
(3) tooth in vitro section chemicals treatment: there is the wheat seedling of scab in (1) in selecting step, will be in the identical different basins of growing way
The interlude with obvious scab as tooth in vitro section, be placed on the filter paper in the fresh-keeping culture medium of drug containing described in step (2),
The both ends of the tooth in vitro section are fixed, then is placed in insulating box and cultivates；
(4) it detects: measuring sorus accounting using photoshop, calculate control efficiency and EC50。
2. method of a kind of measurement fungicide to wheat stripe rust virulence according to claim 1, which is characterized in that step
(1) preparation method of wheat stripe rust spore suspension described in are as follows: wheat stripe rust conidia powder is added to 0.1% polysorbas20
In, continue concussion it is fully dispersed to conidia powder, spore suspension can be prepared.
3. method of a kind of measurement fungicide to wheat stripe rust virulence according to claim 1, which is characterized in that step
(1) seedling described in is the wheat seedling of 1 leaf phase of 1 heart.
4. method of a kind of measurement fungicide to wheat stripe rust virulence according to claim 1, which is characterized in that step
(1) time of the processing of moisturizing described in is that for 24 hours, the condition cultivated in the greenhouse is 17~20 DEG C, illumination 16h/d.
5. method of a kind of measurement fungicide to wheat stripe rust virulence according to claim 1, which is characterized in that step
(1) for the tool used when being inoculated with described in for airbrush, pressure of the airbrush in inoculation is set as 0~0.4bar, described to connect
It is inoculated with when kind according to the amount of every basin wheat seedling 2mL suspension.
6. method of a kind of measurement fungicide to wheat stripe rust virulence according to claim 1, which is characterized in that step
(2) antistaling agent described in includes any one in gibberellin, 6- benzyl aminoadenine or benzimidazole.
7. method of a kind of measurement fungicide to wheat stripe rust virulence according to claim 6, which is characterized in that described red
The mass volume ratio of mycin and water agar is 10~30:1, and the mass volume ratio of the 6- benzyl aminoadenine and water agar is 25
The mass volume ratio of~100:1, the benzimidazole and water agar is 50~100:1, and the unit of the quality volume is μ g/
mL。
8. method of a kind of measurement fungicide to wheat stripe rust virulence according to claim 7, which is characterized in that step
(3) fungicide described in is triazolone, and the ratio between the triazolone and water agar are 0.03~21.87:1, μ g:mL.
9. method of a kind of measurement fungicide to wheat stripe rust virulence according to claim 1, which is characterized in that the side
Method includes the following steps:
(1) inoculated and cultured: wheat stripe rust conidia powder being added in 0.1% polysorbas20, clear water is added after concussion, continues to shake
It swings fully dispersed to conidia powder, spore suspension can be prepared, the quality volume of conidia powder described in the spore suspension is dense
Degree is 5~10 μ g/mL；
(2) it prepares fresh-keeping culture medium: 6- benzyl aminoadenine and triazolone being added in 0.5% water agar and are uniformly mixed,
It pouring into culture dish, the mass volume ratio of the 6- benzyl aminoadenine, triazolone and water agar is 75:0.03~21.87:1,
μg:μg:mL；
(3) circular filter paper of 9cm: being diametrically cut into three sections of 2.5cm, 4.0cm, 2.5cm by tooth in vitro section chemicals treatment first,
In the culture dish that the filter paper item paving of intermediate 4cm is added in step (2)；Next, which takes in step (1), there is the wheat seedling of scab
The wheat of the different basins of the identical sustained height of growing way is chosen in potting, and the conduct with obvious scab of 5cm is in vitro among clip
Leaf section both ends are fixed with remaining 2.5cm filter paper on the filter paper of 4cm and are allowed to be tightly attached to culture base table by leaf section, parallel laid
Face makes containing 5 leaf sections from different basins in each culture dish, then culture dish is placed in illumination in 12 DEG C of constant incubators and is trained
It supports 18h and forms test group；
(4) control group: as a control group with not adding of germicide, other conditions and the identical culture dish of test group；
(5) it detects: carrying out scab accounting pixel analysis with Photoshop software, first record the tooth in vitro of test group and control group
Total pixel of section, re-records total pixel of sorus area in tooth in vitro section；Total pixel of sorus area is calculated with Excel
Average value and sorus area accounting.
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