CN100394182C - Method for measuring virulence of bactericide to bacterium of downy mildew of cucumber - Google Patents
Method for measuring virulence of bactericide to bacterium of downy mildew of cucumber Download PDFInfo
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Abstract
The present invention discloses a method for measuring the virulence of a bactericide to the bacteria of downy mildew of cucumbers, which comprises the following steps: under an aseptic condition, healthy separation leaves on the third leaf bits to the fifth leaf bits of a sterilized cucumber plant are prepared into leaf dishes which are divided into a plurality of groups and are respectively soaked into the diluted bactericide with different concentrations for one to two hours, wherein 0.003 to 0.01% of tween 20 and 0.1 to 0.3 % of methanol which have the same mass percentage concentration are added to the bactericide; the back of each leaf dish is inoculated with the sporangium suspension of the downy mildew of the cucumber, and the sporangium suspension has the concentration of 1.0*10<4> to 1.0*10<5> sporangium /ml; next, each leaf dish is cultured in a wet keeping mode for 20 to 28 hours under dark condition and at the temperature of 17 to 20 DEG C, and then the leaf dish is cultured in a wet keeping mode for five to seven days under the condition that the temperature is from 17 to 20 DEG C, the humidity RH is greater than 80%, and the light is given alternately with dark for 10 to 14 hours; the disease areas on the leaf dishes are measured, and the control efficiency and the EC50 value are calculated. The method has potential social and economic significances.
Description
Technical field
The present invention relates to a kind of method of measuring germifuge to pathogen virulence in the plant protection field, particularly relate to a kind of method of measuring germifuge to the bacterium of downy mildew of cucumber virulence.
Background technology
Cucumber is the main varieties of plant of China's vegetables producing region open country and the cultivation of protection ground, can plant the whole year in most areas.Downy mildew is that cucumber produce to go up one of most important disease, and all there is generation in the area of all cultivated cucumbers, and it is the heaviest to be injured with northern area especially in China, and outdoor cropping is the heaviest with spring stubble cucumber morbidity, and spring is cultivated then on protection ground or fall ill heavier winter.This disease is mainly propagated by air-flow, have infect frequent, the incubation period is short, the serious characteristics of popular harm, can cause plant all dead in several days, is commonly called as " horse race is done ".Also because of after its morbidity, the mustiness thing of a large amount of puces appears in the cucumber blade back, and is referred to as " black wool disease ".
Owing to cucumber downy mildew has become the key factor that the restriction cucumber produces, adopt different prophylactico-therapeutic measuress such as agricultural methods, cultivation disease-resistant variety, biological control, plant quarantine and chemical prevention to alleviate its harm in the production usually.In many prophylactico-therapeutic measuress, the use of chemical agent is still one of main means of control cucumber downy mildew harm, is bringing into play important effect in agricultural production.Domestic and international many research units and farmingization enterprise attempt formulating and screening the new type bactericide that cucumber downy mildew is had remarkable prevention effect always.Because the main target bacterium bacterium of downy mildew of cucumber of its control is the obligate parasite that air-flow is propagated, international germifuge resistance Action Committee (FRAC) thinks that this pathogen has potential high drug-resistance risk to germifuge.Therefore, must take strict resistance monitoring after new type bactericide comes into operation and control measures is avoided and suspension of pesticide resistance generation, prolong the serviceable life of germifuge.
Study reliable and stable, repeatable strong toxicity test method, be used for screening cucumber downy mildew is had the germifuge of excellent preventive effect and sets up the responsive baseline of pathogen to medicament, monitoring field pathogen changes the susceptibility of medicament, being the prerequisite of fungicidal activity screening and resistance to the action of a drug research and improvement, is the basis of realizing plant disease chemical prevention sustainable development.
At non-obligate parasite, the general monitoring of adopting screening that the toxicity test method that exsomatizes carries out medicament and field pathogen to insecticide sensitivity, promptly the agricultural chemicals that does not depend on the cause of disease host that carries out under laboratory condition itself (former medicine) is to the determination method of the bacteriostasis of target bacterium, as spore germination method in traditional toxicity test method and growth rate determination method etc.But bacterium of downy mildew of cucumber is an obligate parasite, and mycelia mainly in the expansion of host cell gap, can not grow on synthetic medium after its spore germination; Simultaneously the pathogen bacterial strain is difficult to carry out long preservation under isolated condition, and several germifuge that are used for the control of cucumber downy mildew evil in producing are distinguished to some extent to the influence of pathogen different developmental phases: as flumorph and dimethomorph to bacterium of downy mildew of cucumber stop that born of the same parents sprout, germ tube elongation and mycelia expansion, sporangiophore be formed with influence; Frost urea cyanogen forms the not influence of each stage to the expansion of bacterium of downy mildew of cucumber mycelia, sporangiophore, and born of the same parents sprout to stopping, the germ tube elongation is influential; Born of the same parents sprout Mancozeb to stopping, the germ tube elongation all has inhibiting effect, and generation, mycelia expansion and the sporangiophore of appresorium formed not influence; The nitrile Fluoxastrobin is except that the generation of sporangiophore, the born of the same parents of stopping are sprouted the not influence, and is all influential to other each stage.Therefore traditional toxicity test method such as spore germination method and growth rate determination method are difficult to detect the true bacteriostasis of this class medicament.At present, generally adopt live body plant method and leaf dish floating method to carry out the toxicity test of germifuge.Live body plant method is used for toxicity test, generally be on the plant of field or greenhouse natural occurrence, to carry out, adopt spray-on process to carry out the germifuge test of different gradient concentrations, general 7-10d spray medicine once, spray altogether three times, the lesion area of plant leaf is scaled disease index before and after the investigation spray medicine, calculates the protection effect of each chemicals treatment under the test inoculation condition.And to convert probit value to the diseases prevention effect data be ordinate, is horizontal ordinate with the drug concentration logarithm, maps and ask its toxicity regression formula, calculates the EC of each medicament to pathogen
50, the virulence of more different medicaments and bacteriostatic activity.It is long that this method exists the mensuration cycle, need be greater than the 40d time; Influence factor is more in the mensuration process, and except that the virulence of medicament itself worked, other various conditions all exerted an influence to the performance of medicament virulence, comprise concrete formulation, dispenser quality, plant growth situation and controlling object fertility condition; Test is repeatable relatively poor, adopts artificial multiple spot investigation method unavoidably to have certain error, has influenced the accuracy and the precision of method; Because relate to greenhouse pot culture or field test, workload was big, need many drawbacks such as consumptive material is more during test was implemented.Live body plant method is used to monitor the susceptibility variation of field pathogen to medicament, and the bacterial strain artificial infection that generally needs to pick up from the field is treated to measure according to the method described above after the plant leaf morbidity to cucumber plant.
Leaf dish floating method is at the drawback in the live body plant method, and the method for a kind of improvement of adopting.Promptly make the leaf dish that diameter is 1.5cm with the card punch cucumber leaves that healthy growth is intact, the floating 5-10 sheet leaf dish of (Ф 9cm) difference in the double dish that contains the contrast of series concentration germifuge and clear water, the bacterium of downy mildew of cucumber spore suspension of inoculation 10 μ L on each leaf dish, keep suspended state, place under the 16-20 ℃ of condition light dark alternately to cultivate 6-8d, check the disease index of each leaf dish, calculate preventive effect, ask its toxicity regression formula and EC
50
Though this method some scholars is at present quoted mutually, but the technician finds to exist cucumber leaf age disunity in practical measurement, pathogen inoculum concentration is inconsistent, flotation fluid is solvent with water, plate-out ability and poor permeability, flotation time long (reaching 6-8d), influence the normal physiological metabolism of blade, the volatile influence factors such as change that cause concentration of treatment of flotation fluid, and finally cause test repeatability and accuracy relatively poor, so different personnel, the measurement result of different batches does not have comparability and quotability yet, can not be with leaf dish floating method as a kind of standard method of germifuge to the bacterium of downy mildew of cucumber toxicity test.
Agriculture chemical examination department of country and medicament research and development department press for a kind of accurate and effective method that detects fungicidal activity of setting up, determining and fungistatic effect and the virulence of more different medicaments, and provide theoretical method for the screening of new type bactericide and the monitoring of field resistance bacterial strain to bacterium of downy mildew of cucumber.
Summary of the invention
The purpose of this invention is to provide a kind of method of measuring germifuge to the bacterium of downy mildew of cucumber virulence.
Mensuration germifuge provided by the present invention is to the method for bacterium of downy mildew of cucumber virulence, be under aseptic condition, make the leaf dish with the healthy excised leaf on the cucumber plant 3-5 leaf position of sterilization, be divided into some groups, for soaking 1-2h in the germifuge of some concentration, be added with the polysorbas20 of the identical 0003-0.01% of mass percentage concentration and the methyl alcohol of 0.1-0.3% respectively at dilution in the described germifuge; Be 1.0 * 10 with each leaf dish back side inoculum density again
4-1.0 * 10
5The sporangia suspension of the bacterium of downy mildew of cucumber of individual sporangium/mL, then it is preserved moisture under 17-20 ℃ of dark condition and cultivate 20-28h, preserve moisture greater than 80%, under the 10-14h alternation of light and darkness condition in 17-20 ℃, humidity RH again and cultivate 5-7d, measure the onset area on the leaf dish, calculate prevention effect and EC
50Value.
In the said method, described healthy excised leaf is preferably the blade on cucumber plant the 4th leaf position; The leaf dish is of a size of ф 1.2-2.0cm, is preferably ф 1.5cm; The content of polysorbas20 is preferably 0.005% in the described germifuge, and the content of methyl alcohol is preferably 0.1%; Described soak time is preferably 1h; The concentration of the sporangia suspension of bacterium of downy mildew of cucumber is preferably 1.0 * 10
4Individual sporangium/mL; The volume of the sporangia suspension that each leaf dish back side drips is 10 μ L; The condition of culture that the back side drips the leaf dish of the sporangia suspension that bacterium of downy mildew of cucumber is arranged is preferably to preserve moisture under 19 ℃ dark condition earlier cultivates 24h, preserves moisture greater than 80%, under the 12h alternation of light and darkness condition in 19 ℃, humidity RH and cultivates 5d.
Need in the immersion process often to stir, to guarantee that each leaf dish is all by moistening uniformly.
Inoculation method is for to blot the soup on the leaf dish, and blade back places training sample ware interior with on the wetting thieving paper of identical drug concentration up, with sessile drop method sporangia suspension is dripped in leaf dish central authorities then.
Used instrument can be the liquid-transfering gun of 5-40 μ L during described inoculating spores suspending liquid.
The present invention is directed to present bacterium of downy mildew of cucumber (obligate parasite) bacterial strain can not long preservation, and the live body plant method of employing is measured drawbacks such as the experimental period that exists in the toxicity action process of germifuge to bacterium of downy mildew of cucumber is long, and influence factor is many, and workload is big; Uncertain at the cucumber leaf age/leaf position and the pathogen inoculum concentration that exist in the leaf dish floating method mensuration process, flotation fluid is solvent with water, plate-out ability and poor permeability, flotation time long (reaching 6-8 days) in liquid solution, influence the normal physiological metabolism of blade, the easy chrysanthemum of leaf dish and rotten causes error occurring in the lesion area investigation, the problem of influence such as the volatile change that causes concentration of treatment of flotation fluid test repeatability and accuracy.Set up the assay method of the high detection germifuge of reliable and stable, good reproducibility, precision to the bacterium of downy mildew of cucumber virulence, with its called after leaf dish cultivation of preserving moisture, this method mensuration cycle is about 5 days; Reagent agent is the former medicine of high-load in the mensuration process, is not subjected to the influence of application method and formulation; Former medicine has strengthened soup opening up and permeate on the leaf dish by the quantitative interpolation of surperficial spreader-sticker and organic solvent, and leaf dish floating time in soup shortens to 1h, stopped the leaf dish chrysanthemum phenomenon that suspends and cause in soup because of long-time; The mensuration process is carried out on stripped leaf dish, be not depend on agricultural chemicals (former medicine) that the live body plant carries out mensuration itself for the bacteriostasis of target bacterium, be not subjected to the influence of plant growing state, the accuracy of method and precision height, collimation is good, the examination material amount that expends in the testing process is few, can measure germifuge accurately, reliably to the variation of the toxicity action of bacterium of downy mildew of cucumber and monitoring field bacterium of downy mildew of cucumber, for further germifuge mechanism of action and resistance to the action of a drug research and improvement provide technical support to fungicide sensitivity.The present invention can be used as a kind of new method with standard feature, is used to measure the toxicity action and monitoring field bacterium of downy mildew of cucumber variation to fungicide sensitivity of germifuge to bacterium of downy mildew of cucumber, has potential social and economic significance.
Description of drawings
Fig. 1 measures the virulence of germifuge to bacterium of downy mildew of cucumber for the cultivation of preserving moisture with the leaf dish
Fig. 2 is the incidence that the germifuge of variable concentrations is handled the posterior lobe dish
Fig. 3 is the susceptibility distribution of bacterium of downy mildew of cucumber to flumorph
Fig. 4 is the susceptibility frequency distribution of 77 strain bacterium of downy mildew of cucumber to flumorph
Embodiment
Method therefor is conventional method if no special instructions among the following embodiment.
Reagent agent:
The former medicine of 96% flumorph (flumorph) (Shenyang Chemical Engineering Inst); The former medicine of 97% metalaxyl (metalaxyl) (Singapore Li Nong private limited partnership); 95% nitrile Fluoxastrobin (azoxystrobin) suspending agent (just reaching earlier China Investment Ltd.).
The test plant material:
Susceptible cucumber variety Chang Chun Mi Ci is cultivated the 15cm in ф, in the pot for growing seedlings of high 12cm, place glasshouse to cultivate, treat that plant is long to gather the blade of identical leaf position, be used for the cultivation and the toxicity test of bacterium of downy mildew of cucumber to 10 leaf after dates.
1, the leaf dish selection of cucumber leaf age in the determination method of preserving moisture
Healthy leaves on 3-10 the leaf position of collection cucumber plant, with sterile water wash 3 times, respectively the blade on the various position leaves is made the leaf dish with the card punch of ф 15mm then, what blade back was neat up is positioned in the ф 150mm double dish of preserving moisture with thieving paper, 50 leaf dish of every ware (Fig. 1) are 1 * 10 in each leaf dish central authorities inoculation 10 μ L concentration
4Sporangium/sporangial the suspending liquid of mL bacterium of downy mildew of cucumber, and placing 20 ℃, RH＞80% is cultivated between the growth of 12h alternation of light and darkness, mould aspect on the 7d " Invest, Then Investigate " leaf dish is long-pending, the severity of bacterium of downy mildew of cucumber morbidity (test repeats 3 times) on the more different leaf age cucumber leaf dish.The result is as shown in table 1, showing that blade on the various position leaves infects bacterium of downy mildew of cucumber has a significant effect, 3rd, onset area does not have significant difference between the leaf dish made of 4,5 and 6 leaf position blades, and the leaf dish onset area that the 7th, 8,9,10 leaf position blades are made obviously reduces, show the increase along with leaf age, blade has certain resistivity to the intrusion of bacterium of downy mildew of cucumber.Therefore, select blade on the 4th leaf position to carry out the sensitivity testing of medicament, can reduce the error that causes by the difference between the blade, also can save and cultivate the time that vegetable material needs.
The influence that table 1 cucumber leaves leaf age infects bacterium of downy mildew of cucumber
Annotate:
a: 3 first leaf representing plant to launch fully, along with numerical value increases, also the phase strain is big for leaf age.
b: the standard error between three repetitions.
2, the leaf dish selection of inoculum concentration in the determination method of preserving moisture
Gather the 4th blade of cucumber plant, 1 method is made the leaf dish set by step, and processing of per 50 leaf dish is inoculated 10 μ L concentration with sessile drop method in each leaf dish back side and is respectively 1 * 10
3, 1 * 10
4With 1 * 10
5The suspending liquid of individual sporangium/mL, the cultivation of preserving moisture then, the onset area of measurement leaf dish behind the 7d, relatively the different vaccination bulk concentration is to the influence (test repeats 3 times) of morbidity.The result adds up onset area as shown in Figure 2, and statistics is as shown in table 2, and inoculum density is 1 * 10
4With 1 * 10
5Do not have significant difference between the leaf dish onset area of the suspending liquid of individual sporangium/mL, and inoculum density is 1 * 10
3The leaf dish morbidity severity of individual sporangium/mL obviously reduces, and the surface seeding bulk concentration also can exert an influence to the order of severity of morbidity, therefore, selects 1 * 10
4The concentration of individual sporangium/mL is inoculated.
The influence that table 2 inoculum concentration infects bacterium of downy mildew of cucumber
3, preserve moisture surperficial spreader-sticker and choice of Solvent in the determination method of leaf dish
The polysorbas20 of mensuration 0.005% and 0.1% methanol solvate are contrast to the influence of measurement result with the sterilized water.Gather the 4th blade of cucumber plant, 1 method is made the leaf dish set by step, processing of per 50 leaf dish, respectively at the 100mL sterilized water, 0.005% polysorbas20 solution, 0.1% methanol solution and contain 0.005% polysorbas20 simultaneously and the solution of 0.1% methyl alcohol in soak 1h, often stir in the immersion process, to guarantee that each leaf dish is all by wetting uniformly.1 method connects bacterium, cultivation and data processing set by step then, comparison surface spreader-sticker and organic solvent are to the influence of morbidity, the result is as shown in table 3,0.005% polysorbas20 and 0.1% methyl alcohol all influence less than obvious test findings, show and to adopt concentration to be lower than 0.005% polysorbas20 as surperficial spreader-sticker, 0.1% methyl alcohol (methanol) carries out the dissolving of former medicine as organic solvent, and the content of methyl alcohol in the medicament dilution is controlled at≤0.1% scope in.
The influence that surperficial spreader-sticker of table 3 (Tween 20) and organic solvent (methyl alcohol) infect bacterium of downy mildew of cucumber
4, the leaf dish precision test of determination method of preserving moisture
Select 6 bacterial strain of cucumber downy mildew bacteria at random for use, adopt the leaf dish method of preserving moisture to carry out the germifuge flumorph to its toxicity test, concrete grammar is: get the healthy excised leaf of the 4th leaf position on the cucumber plant, make the cucumber leaf dish of ф 15mm.Mixing is divided into 7 groups at random, and every group of 50 leaf dish soak 1h in the flumorph soup respectively at series concentration (0.00,0.05,0.10,0.30,0.50,0.70 and 1.00 μ g/mL).Often stir in the immersion process, to guarantee that each leaf dish is all by moistening uniformly.Each is handled and all to contain 0.005% polysorbas20 and 0.1% methyl alcohol.After soaking end the soup on the leaf dish is blotted, blade back places training sample ware interior with on the wetting thieving paper of identical drug concentration up, is every milliliter with the sessile drop method inoculum density then and contains 10
4Individual sporangial suspending liquid 10 μ L are in leaf dish central authorities.Postvaccinal leaf dish is positioned under 19 ℃, the condition of RH＞80%, 12h alternation of light and darkness cultivates, measure the onset area on the leaf dish behind the 5d, calculate prevention effect and EC
50Each bacterial strain repeats 5 times, calculates the EC of medicament to each bacterial strain
50And the coefficient of variation between 5 repetitions of each bacterial strain, by the precision of coefficient of variation confirmed test method.6 strains testeds, the precision measurement result that each bacterial strain repeats for 5 times is as shown in table 4, and the coefficient of variation between 5 repetitions has all shown higher repeatability between 1.4-5.3%.Further the proof leaf dish determination method of preserving moisture has good repeatability and precision, and this method is applicable to carries out the mensuration of germifuge flumorph to obligate parasite bacterium of downy mildew of cucumber virulence.
The table 4 leaf dish determination method of preserving moisture is used for the quick repeatability to the bacterium of downy mildew of cucumber toxicity test of flumorph
5, the mutual resistant test between the germifuge
Adopt the assay method in the above-mentioned precision test, select for use 6 strain metalaxyls and nitrile Fluoxastrobin drug-fast strain to carry out mutual resistant test between flumorph, metalaxyl and the nitrile Fluoxastrobin respectively, the result is as shown in table 5, and metalaxyl and nitrile Fluoxastrobin are to the EC of metalaxyl drug-fast strain and nitrile Fluoxastrobin drug-fast strain
50Respectively between 63.10-87.10 μ g/mL and 2.82-7.33 μ g/mL, and flumorph is to the EC of all bacterial strains
50All between 0.08-0.18 μ g/mL, statistical results show: flumorph and metalaxyl (r=0.000685, n=6) and flumorph and nitrile Fluoxastrobin (r=0.220137, n=6) correlativity between is all very low, proves between flumorph and metalaxyl and the nitrile Fluoxastrobin not have the mutual resistance to the action of a drug.
Table 56 strain metalaxyl drug-fast strain and 6 strain nitrile Fluoxastrobin drug-fast strains are to the susceptibility of flumorph, metalaxyl and nitrile Fluoxastrobin
Embodiment 2, the leaf dish method of preserving moisture is measured the virulence (monitoring of field drug-fastness bacterial strain of germifuge to bacterium of downy mildew of cucumber
Reagent agent:
The former medicine of 96% flumorph (flumorph) (Shenyang Chemical Engineering Inst).
For trying plant:
Susceptible cucumber variety Chang Chun Mi Ci is cultivated the 15cm in ф, in the pot for growing seedlings of high 12cm, place glasshouse to cultivate, treat that plant is long to gather the blade of the 4th leaf position, be used for the cultivation and the toxicity test of bacterium of downy mildew of cucumber to 6 leaf after dates.
Strains tested:
Gather the cucumber downy mildew sample from the cucumber cultivation ground that flumorph and similar medicament dimethomorph were not used in Dong Gaocun Cui village Technology Park, Pinggu, Wang Huacun western food ground, Huairou, Kong Huaying village, Yongning, Yanqing town, Hao Zhuan apple garden, south, Changping and the areas such as China Agricultural University's Changping experiment centre, beans shop, Fangshan village, northwest, Haidian District Wang Cun and Hebei mountains in a range are flat, Inner Mongol of Beijing suburb, separate to obtain 77 strain sensitive strains, bacterial strain is carried out that subculture is cultivated and deepfreeze is preserved for a long time according to the method among the embodiment 1.
Measure the susceptibility of bacterium of downy mildew of cucumber according to the method for preserving moisture of the leaf dish among the embodiment 1 to germifuge.Concrete grammar is: gather cucumber plant the 4th leaf position blade, the leaf dish of preparation ф 15mm, mixing at random, be divided into 7 groups, every group of 50 leaf dish, divide to soak in the 1h immersion process in small pin for the case 0,0.05,0.10,0.30,0.50,0.70, the 1.000 μ g/mL flumorph solution and often stir, to guarantee that each leaf dish is all by moistening uniformly.Each is handled and all to contain 0.005% polysorbas20 and 0.1% methyl alcohol.After soaking end the soup on the leaf dish is blotted, blade back places up with on the wetting thieving paper of identical drug concentration, is every milliliter with the sessile drop method inoculum density then and contains 10
4Individual sporangial suspending liquid 10 μ L are in leaf dish central authorities.Postvaccinal leaf dish is positioned under 19 ℃, the condition of RH＞80%, 12h alternation of light and darkness cultivates, measure the onset area on the leaf dish behind the 7d, calculate prevention effect and EC
50Measure 77 bacterial strains gathering from the field susceptibility with this method, set up responsive baseline, measure as shown in Figure 3, wherein EC flumorph
50That maximum is 0.2647 μ g/mL, and that minimum is 0.0213 μ g/mL, differs 12.43 times, average EC
50=(0.1360 ± 0.0487) μ g/mL.From 77 strain bacterium of downy mildew of cucumber as can be seen to the susceptibility histogram (Fig. 4) of flumorph, 77 bacterial strains distribute to the susceptibility of flumorph and are continuous unimodal curve, the anti-medicine cause of disease subgroup that susceptibility descends do not occur, therefore available this unimodal curve is as the responsive baseline that carries out in the future the field drug-fastness monitoring.
Claims (8)
1. method of measuring germifuge to the bacterium of downy mildew of cucumber virulence, may further comprise the steps: 1) under aseptic condition, make the leaf dish with the healthy excised leaf on the cucumber plant 3-5 leaf position of sterilization, be divided into some groups, for soaking 1-2h in the germifuge of some concentration, be added with the polysorbas20 of the identical 0.003-0.01% of mass percentage concentration and the methyl alcohol of 0.1-0.3% respectively at dilution in the described germifuge; 2) each leaf dish back side is placed up on the thieving paper in the double dish, described thieving paper is used with to soak this leaf dish same concentrations germifuge wetting; Is 1.0 * 10 with sessile drop method with concentration
4-1.0 * 10
5The sporangia suspension of the bacterium of downy mildew of cucumber of individual sporangium/mL drips in leaf dish central authorities; 3) the leaf dish is preserved moisture under 17-20 ℃ of dark condition and is cultivated 20-28h, preserves moisture greater than 80%, under the 10-14h alternation of light and darkness condition in 17-20 ℃, humidity RH and cultivates 5-7d, measures the onset area on the leaf dish, calculates prevention effect and EC
50Value.
2. method according to claim 1 is characterized in that: described healthy excised leaf is the blade on cucumber plant the 4th leaf position.
3. method according to claim 1 is characterized in that: the content of polysorbas20 is 0.005% in the described germifuge, and the content of methyl alcohol is 0.1%.
4. method according to claim 1 is characterized in that: the concentration of the sporangia suspension of described bacterium of downy mildew of cucumber is 1.0 * 10
4Individual sporangium/mL.
5. according to claim 1 or 2 or 3 or 4 described methods, it is characterized in that: described leaf dish is of a size of φ 1.2-2.0cm.
6. method according to claim 5 is characterized in that: described leaf dish is of a size of φ 1.5cm; The volume of the sporangia suspension that each leaf dish back side drips is 10 μ L.
7. according to claim 1 or 2 or 3 or 4 described methods, it is characterized in that: the described back side drips the condition of culture of the leaf dish of the sporangia suspension that bacterium of downy mildew of cucumber is arranged and cultivate 24h for preserving moisture earlier under 19 ℃ dark condition, preserves moisture greater than 80%, under the 12h alternation of light and darkness condition in 19 ℃, humidity RH and cultivates 7 days.
8. according to claim 1 or 2 or 3 or 4 described methods, it is characterized in that: constantly stir in the described immersion process.
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| CN102827917A (en) * | 2012-09-04 | 2012-12-19 | 青岛农业大学 | Method for measuring toxicity of sterilizing agent on brassica rapa pekinensis peronospora parasitica |
| CN103884825B (en) * | 2014-03-29 | 2015-08-05 | 广西大学 | A kind of evaluation of pesticide effectiveness method of preventing and treating sheath and culm blight of rice medicine |
| CN110146668A (en) * | 2019-05-21 | 2019-08-20 | 西南大学 | A method of measurement fungicide is to wheat stripe rust virulence |
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