CN102754655B - Medium for simple, convenient and quick pythium aphanidermatum isolation - Google Patents
Medium for simple, convenient and quick pythium aphanidermatum isolation Download PDFInfo
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- CN102754655B CN102754655B CN201210250623.5A CN201210250623A CN102754655B CN 102754655 B CN102754655 B CN 102754655B CN 201210250623 A CN201210250623 A CN 201210250623A CN 102754655 B CN102754655 B CN 102754655B
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- 238000002955 isolation Methods 0.000 title abstract description 5
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Abstract
The invention discloses a selective medium for simply, conveniently and quickly isolating pythium aphanidermatum, which is particularly suitable for isolation of pythium aphanidermatum, and belongs to the technical field of microbes. The medium is obtained through adding the following disinfectant (1) or (2) into a PDA (Potato Dextrose Agar) medium: (1) is a disinfectant consisting of carbendazol, pentachloronitrobenzene, flumorph and rifampicin; and (2) is a disinfectant consisting of prochloraz, pentachloronitrobenzene, flumorph and rifampicin. Experiments show that the PDA selective medium has the characteristics of feasibility, accuracy, practicability, quickness, low cost and simplicity and convenience in preparation. When the PDA selective medium is used for isolating pythium aphanidermatum and other pythium oomycetes, the problem of serious contamination of various non-target fungus and bacteria in the isolation process of pythium aphanidermatum and other plant pathogenic pythium is solved, and the effects of quickness, high efficiency and labor and time conservation are achieved.
Description
Technical field
The present invention relates to a kind of selective medium of easy, quick separated pythium spp.Belong to microbial technology field.
Background technology
Pythium spp is universal soil fungi, saprophytic very capable, can infect root and the seedling of 150 various vegetables fruit crops, causes rotten kind, dampings off, stands withered, rotten and decayed fruit.Wherein, the damping off being caused by melon and fruit corruption mould (Pythium aphanidermatum) is Young Plant a kind of worldwide destructive diseases in seedling stage such as melon dish, flowers, medicinal plant, tobacco and beans, cotton, corn, Chinese sorghum.By conventionally after emerging, basal part of stem place near the ground produces the dark-coloured spot of water stain shape, and then around stem expansion, hanging gradually contracting is thin-line-shaped, and seedling overground part is rolled over down because losing enabling capabilities, is commonly called as the disease of seizing by the throat.During high humidity, disease portion or soil table can produce white cotton floccule, i.e. germ mycelia, sporangiophore and sporangium.Under the condition of benign climate, just can break out in a short time, rate of propagation is exceedingly fast, and in serious situation, can cause plant seedlings Large Scale Death, and causing is short of seedling even ruins kind, to agricultural production, brings serious economic loss.
Melon and fruit corruption mould (P.aphanidermatum) belongs to oomycetes door Peronosporales pythiaceae pythium.Germ survives the winter at 12-18cm topsoil with egg spore, and survival for a long time in soil.The primary source of infection of damping off is mainly survived the winter in soil, invalid body with rotten mould egg spore and the chlamydospore of melon and fruit, and wherein the invalid body bacterial bearing rate in soil is the highest.Can sprout generation sporangium under optimum conditions, with zoospore or directly grow germ tube and infect seedling and cause and damping off.Infecting again of field is main by output Sporangia and zoospores on sick seedling, borrows irrigation water or rainwater to spatter and on the rhizome that is attached to ground proximity, causes more serious loss.This disease is mainly after coming up, occur before growing 1-2 sheet leaf.Especially there is low temperature, super-humid conditions in nursery stage, is beneficial to morbidity.
For to the rotten mould correlative study of carrying out of melon and fruit, and the field incidence of investigation pathogen, screen efficient medicament, finally reach the object of effective this disease of control, the separated melon and fruit that obtains purifying rotten mould be necessary precondition.
Conventional pathogenicbacteria separation method is that < < plants use 75% ethanol and the 0.1% mercuric chloride liquid surface sterilization processing that disease research method > > introduces.In real work, the sample collecting from field is except by rotten mould the infecting of melon and fruit, inevitably usually with sickle-like bacteria, other non-target fungus and bacteriums such as Rhizoctonia solani Kuhn and epidemic disease are mould, while carrying out chorista pathogen according to a conventional method, due to the pollution of these non-target fungies or bacterium, cause object bacteria separation rate low or be difficult to be separated to object bacteria.Therefore, the separated melon and fruit that utilizes ordinary culture medium diseased tissues or in soil is rotten when mould, does not reach the object of separation and purification.
Summary of the invention
An object of the present invention is to provide a kind of bactericide combination.
Bactericide provided by the present invention combination, its active component is as following 1), 2) as shown in:
1), by carbendazim, pcnb, flumorph, rifampin, formed;
2), by Prochloraz, pcnb, flumorph, rifampin, formed;
In above-mentioned bactericide,
Described 1) in, the ratio of quality and the number of copies of described carbendazim, pcnb, flumorph and rifampin is (10-50): (10-50): (5-10): (50-100), be specially 10: 10: 5: 50 or 20: 20: 7: 70 or 50: 50: 10: 100;
Described 2) in, the ratio of quality and the number of copies of described Prochloraz, pcnb, flumorph and rifampin is (10-20): (10-50): (5-10): (50-100), be specially 10: 10: 5: 50 or 15: 20: 7: 70 or 20: 50: 10: 100;
Another object of the present invention is to provide a kind of medium for separating of melon and fruit corruption mould (P.aphanidermatum).
Medium for separating of melon and fruit corruption mould (P.aphanidermatum) provided by the present invention, shown in following I or II:
I, to adding described in claim 1 or 21 in PDA medium) shown in bactericide obtain;
II, to adding described in claim 1 or 22 in PDA medium) shown in bactericide obtain;
In above-mentioned medium, shown in described I, in medium, the concentration of described carbendazim in described PDA medium is 10 μ g/mL-50 μ g/mL, is specially 10 μ g/mL, 20 μ g/mL or 50 μ g/mL; The concentration of described pcnb in described PDA medium is 10 μ g/mL-50 μ g/mL, is specially 10 μ g/mL, 20 μ g/mL or 50 μ g/mL; The concentration of described flumorph in described PDA medium is 5 μ g/mL-10 μ g/mL, is specially 5 μ g/mL, 7 μ g/mL or 10 μ g/mL; The concentration of described rifampin in described PDA medium is 50 μ g/mL-100 μ g/mL, is specially 50 μ g/mL, 70 μ g/mL or 100 μ g/mL;
In above-mentioned medium, shown in described II, in medium, the concentration of described Prochloraz in described PDA medium is 10 μ g/mL 20 μ g/mL, is specially 10 μ g/mL, 15 μ g/mL or 20 μ g/mL; The concentration of described pcnb in described PDA medium is 10 μ g/mL-50 μ g/mL, is specially 10 μ g/mL, 20 μ g/mL or 50 μ g/mL; The concentration of described flumorph in described PDA medium is 5 μ g/mL-10 μ g/mL, is specially 5 μ g/mL, 7 μ g/mL or 10 μ g/mL; The concentration of described rifampin in described PDA medium is 50 μ g/mL-100 μ g/mL, is specially 50 μ g/mL, 70 μ g/mL or 100 μ g/mL;
The application of above-mentioned medium in separated melon and fruit corruption mould (P.aphanidermatum) also belongs to protection scope of the present invention.
The method of separated melon and fruit corruption provided by the present invention mould (P.aphanidermatum), comprise the steps: to carry out separation with above-mentioned arbitrary described medium, for separating of sample for infecting rhizome or the soil of the plant of melon and fruit corruption mould (P.aphanidermatum).
Experiment showed, PDA selective medium of the present invention have feasibility, accuracy, practical quick and with low cost, prepare easy feature.Mould with the separated melon and fruit corruption of PDA selective medium of the present invention, can solve that melon and fruit is rotten mouldly waits the with serious pollution problem of various non-target fungus and bacterium in the rotten mould separation process of pathogenic, and speed is fast, efficiency is high, saves time, laborsaving.The present invention provides reference for separated other pythium bacterial strains of success, for the rotten mould technical support that provides of the disposable separation in field and indoor purifying melon and fruit is provided.The present invention select medium can be used for easy, from diseased plant tissue or sick field soil, disposable separated melon and fruit is rotten mould and the corruption of indoor purifying melon and fruit is mould quickly.
Accompanying drawing explanation
Fig. 1 is the inhibitory action of three kinds of antibiotic to plant rhizome surface bacteria.
Fig. 2 is the inhibitory action of three kinds of antibiotic to bacterium in soil.
Fig. 3 is incidence and the separation case of the rotten mildew of melon and fruit.A is healthy cucumber seedling; B is the sick seedling of the rotten mould damping off causing of melon and fruit; C is the sick seedling of the rotten mould damping off causing of field melon and fruit; D is the rotten mould fusarium wilt in the rotten mould lawn causing of melon and fruit; E is the rotten mould damping off causing of stage melon and fruit of growing seedlings; F is that the melon and fruit corruption being separated in disease plant is mould.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Carbendazim active compound is purchased from Guangxin, Anhui Nong Hua limited company (Guangxin, Anhui Nong Hua Group Co.,Ltd), the chemical name of its main active material is N-(2-benzimidazole base)-methyl carbamate, adopted name is carbendazim, molecular formula: C9H9N3O2.In this medicine, the quality percentage composition of main active material is 98.9%.
The former medicine of pcnb is purchased from Shanxi San Li Chemical Co., Ltd. (former Shanxi Province Linfen Organic Chemical Plant), and the chemical name of its main active material is pcnb, and adopted name is pcnb, molecular formula: C6Cl5NO2.In this medicine, the quality percentage composition of pcnb is 95%.
The former medicine of Prochloraz (being again " prochloraz ") is purchased from Hainan Li Zhi biotechnology Co., Ltd, the chemical name of its main active material is N-propyl group-N-[2-(2,4,6-Trichlorophenoxy) ethyl]-imidazoles-1-formamide, molecular formula: C15H16Cl3N3O2.In this medicine, the quality percentage composition of Prochloraz is 97%.
The former medicine of flumorph is purchased from Shenyang Chemical Engineering Inst, and the chemical name of its main active material is 4-[3-(3,4-dimethoxy)-3-(4-fluorophenyl) acryloyl] morpholine, adopted name is flumorph, molecular formula: C21H22FNO4.In this medicine, the quality percentage composition of main active material is 96%.
Penicillin, rifampin, chloramphenicol are all purchased from Beijing Tuo Yingfang Science and Technology Ltd..
Embodiment 1, three kind of fungistat, the mould inhibitor of a kind of epidemic disease and three kinds of antibiotic are to the rotten mould inhibitory action of melon and fruit
One, three kinds of fungistats and the mould inhibitor of a kind of epidemic disease are to the rotten mould inhibitory action of melon and fruit
The solvent that the dimethyl formamide of take is carbendazim, take the solvent that acetone or methyl alcohol is Prochloraz, pcnb and flumorph, prepares respectively four kinds of medicament mother liquor 10ml of 5000 μ g/mL; Wherein, the mother liquor of these three kinds of fungistats of carbendazim, pcnb and Prochloraz is prepared respectively to the pastille PDA flat board that series concentration is 50 μ g/mL, 20 μ g/mL and 10 μ g/mL; It is that the pastille PDA of 20 μ g/mL, 10 μ g/mL and 5 μ g/mL is dull and stereotyped that the mother liquor of the mould inhibitor of this epidemic disease of flumorph is prepared respectively to series concentration, and the flat board that is added with solvent of take is contrast; Then adopt mycelial growth rate method to measure the rotten mould susceptibility to bacterium spirit, pcnb, Prochloraz and flumorph of melon and fruit.By for examination the strain of melon and fruit pythium spp on PDA flat board in 25 ℃ of preculture 2d, the card punch that is 5mm with diameter is beaten and is got bacterium cake on the same circumference of colony edge, by pure culture biscuits involvng inoculation to the dull and stereotyped central authorities of the PDA pastille that contains respectively four kinds of medicine series concentration (each concentration is containing equivalent organic solvent), dark culturing at 25 ℃, after 2-3d, by right-angled intersection method, measure each bacterium colony of processing and increase diameter (colony diameter deducts bacterium cake diameter 5mm), every processing repeats 3 times; Finally according to formula below, obtain each drug concentration to the inhibition percentage of mycelial growth (%), inhibiting rate (%)=[(contrast bacterium colony increases diameter-processings bacterium colony and increases diameter)/(contrast bacterium colony growth diameter)] * 100.
Measured for four kinds of fungistats that try the rotten mould inhibitory action of melon and fruit, result is as shown in table 1, the carbendazim of variable concentrations, pcnb all lower than 10%, therefore can adopt the carbendazim, pcnb of 10-50 μ g/mL as selective pesticides to the rotten mould inhibiting rate of melon and fruit; The Prochloraz that 50 μ g/mL Prochlorazs are 27%, 10-20 μ g/mL concentration to the rotten mould inhibiting rate of melon and fruit is lower to the rotten mould inhibiting rate of melon and fruit, so can adopt the selective pesticides that the Prochloraz of 10-20 μ g/mL is separated rotten mould middle use.Concentration is that 20 μ g/mL flumorphs are greater than 11.2% to the rotten mould inhibiting rate of melon and fruit, 10 μ g/mL flumorphs are 4.5% to the rotten mould inhibiting rate of melon and fruit, the concentration of 2 μ g/mL does not have inhibitory action to Phytophthora capsici, therefore, can adopt the flumorph of 2-10 μ g/mL concentration as selective pesticides;
Four kinds of fungistats of table 1 are to the rotten mould inhibitory action of melon and fruit
Two, three kinds of antibiotic are to the rotten mould inhibitory action of melon and fruit
With aqua sterilisa, above-mentioned three kinds of antibiotic are all mixed with to 5 * 10
4the mother liquor of μ g/mL, is used three kinds of mother liquors to be mixed with respectively rifampin, penicillin and the chloramphenicol that concentration is 100 μ g/mL, 50 μ g/mL, and the flat board that is added with sterile water of take is contrast.Then adopt mycelial growth rate method to measure melon and fruit rotten mould to these three kinds of antibiotic susceptibility.By for examination the strain of melon and fruit pythium spp on PDA flat board in 25 ℃ of preculture 2d, the card punch that is 5mm with diameter is beaten and is got bacterium cake on the same circumference of colony edge, pure culture biscuits involvng inoculation is dull and stereotyped central to the PDA pastille that contains respectively medicament series concentration, dark culturing at 25 ℃, after 2-3d, by right-angled intersection method, measure each bacterium colony of processing and increase diameter (colony diameter deducts bacterium cake diameter 5mm), every processing repeats 3 times; Finally according to formula below, obtain each drug concentration to the inhibition percentage of mycelial growth (%), inhibiting rate (%)=[(contrast bacterium colony increases diameter-processings bacterium colony and increases diameter)/(contrast bacterium colony growth diameter)] * 100.
Three kinds of antibiotic simultaneously having measured variable concentrations, to the rotten mould inhibitory action of melon and fruit, the results are shown in Table 2, and concentration is that penicillin, rifampin and the chloramphenicol of 100 and 50 μ g/mL is to the rotten mould obvious inhibitory action that all do not have of melon and fruit.Therefore, can when separated melon and fruit corruption is mould, use 100 μ g/mL or penicillin, rifampin and the chloramphenicol of 50 μ g/mL concentration as the antibiotic composition in the mould selective medium of separation and purification melon and fruit corruption.The inhibitory action of the fixed three kinds of antibiotic of the pacing of going forward side by side to the bacterium of carrying with soil bacteria and capsicum tissue in environment.
Three kinds of antibiotic of table 2 are to the rotten mould inhibitory action of melon and fruit
Embodiment 2, the three kind of antibiotic inhibitory action to bacterium in environment
For trying plant: susceptible cucumber variety Chang Chun Mi Ci is cultivated to the 15cm in φ, in the pot for growing seedlings of high 12cm, be placed in China Agricultural University research park capsicum experimental field and cultivate, plant grows to 2 leaf after dates and gathers immediately rhizome portion, for the preparation of bacteria suspension.
For trying soil: in 2011 from the serious area of Daxing District damping off morbidity, gather soil sample, standby.
Test method: this test is to detect selected three kinds of concentration antibiotic whether can effectively suppress the bacterium in environment on the basis of embodiment 1 result.Detection for bacterium in environment, mainly for the bacterium from plants stems base portion and morbidity soil, this be according to the rotten mould separated part of melon and fruit be mainly the basal part of stem of morbidity and in spite of illness soil determine, what this experiment adopted is the soil sample in the more serious field, Daxing District of Cucumis sativus stem base portion and damping off generation.First, adopt the rhizome portion of 30 cucumber seedlings, shred rear employing quartering sample is divided into 4 parts at random, take out wherein 1 part and put into 25ml centrifuge tube, add 15ml aqua sterilisa, 200r/min vibration 2 minutes, crosses leaching filtrate, use sterile water dilution 100 *, get respectively 100 μ l and be coated on rifampin, penicillin and the chloramphenicol that contains respectively 100 μ g/mL, 50 μ g/mL, the not pastille flat board of take is contrast, and 3 repetitions of every processing, observe its inhibitory action to bacterium for 2 days afterwards; Field, Daxing, Beijing soil sampling of then falling ill serious from damping off, take 5g, put into beaker and add 20ml aqua sterilisa, the 200r/min 20min that vibrates, get supernatant 100 μ l and be coated on respectively rifampin, penicillin and the chloramphenicol that contains 100 μ g/mL, 50 μ g/mL, the not pastille flat board of take is contrast, and 3 repetitions of every processing, observe its effect to bacterium for 2 days afterwards.Selection has obvious inhibiting antibiotic concentration to bacterium.
This research, on the basis of table 2 result, has been measured respectively three kinds of antibiotic that initial option can use inhibitory action to bacterium in seedling basal part of stem and soil, and the result of two researchs is consistent.From Fig. 1 and Fig. 2, find out, chloramphenicol bacteriostatic activity is poor, and when its concentration reaches 100 μ g/mL, compared with the control, fungistatic effect is not obvious.The bacteriostatic activity of penicillin, rifampin is stronger, can reach antibacterial effect.Due to the diversity of bacterial species in environment, penicillin mainly has stronger inhibitory action to gram-positive bacteria, so there is a little fragmentary Gram-negative bacteria bacterium colony in flat board.And for rifampin, in flat board, only have a little fungus colony.Therefore, rifampin has the bacteriostasis of wide spectrum to the bacterium in seedling rhizome portion and soil.
In sum, the principle that the present invention screens medicament is that the rotten mould inhibiting rate of melon and fruit is less than under 10% prerequisite, selects as far as possible the medicament of high concentration, to improve the bacteriostatic activity to non-object bacteria.According to above all result of the tests, can find out, be applied to the rotten mould selective medium of disposable separated melon and fruit from field, the fungistat that can select is carbendazim, Prochloraz, pcnb and flumorph, and concentration is respectively 10-50 μ g/mL, 10-20 μ g/mL, 10-50 μ g/mL and 5-10 μ g/mL; The antibiotics that can select is rifampin, and concentration adopts 50-100 μ g/mL.
Embodiment 3, for separating of the rotten mould PDA selective medium of melon and fruit
One, separating method
Between 2011 and 2012, from Daxing, Beijing, Pinggu, Changping, the serious area of Shunyi District damping off morbidity gather sick sample, mould for separating of melon and fruit corruption.
The rotten mould separation of melon and fruit is carried out as follows: in areal, select different fields, the sick sample (rhizome and soil) that gathers damping off carries out separated.
Separating step: use clear water to clean out incidence tissue and fully dry afterwards, the fritter tissue that cuts the sick strong intersection of rhizome portion is seeded on selective medium of the present invention, dark culturing at 25 ℃, to there being mycelia to grow; The eugonic fritter mycelium of picking is to clear water, at 20 ℃, cultivate 24-48h, can be observed mycelia top and generate a large amount of sporangiums, and sporangium 12-24h in water can sprout and produce vesicle, after 15-30min, zoospore can discharge from vesicle; Choose single zoospore and be placed on common PDA medium and cultivate, to growing bacterium colony, obtain melon and fruit corruption mould.To rotten mould evaluation of the melon and fruit obtaining.Real melon and fruit pythium spp is fallen to being kept on PDA test tube slant, add appropriate sterilizing paraffin oil and be placed under room temperature and save backup.
According to existing melon and fruit pythium spp authentication method, identify.
Two, the medium using in separation
(1) 1, the compound method of potato dextrose agar (PDA): potato 200g, glucose 20g, agar powder 14g, adding distil water is settled to 1000mL, high pressure steam sterilization, 121 ℃, 20min.
2, the compound method of corn flour-agar medium (CMA): corn flour 300g, double gauze filters, agar powder 14g, adding distil water is settled to 1000mL, high pressure steam sterilization, 121 ℃, 20min.
(2) from morbidity field soil or sick sample rhizome part, from the different PDA of the rotten mould use of melon and fruit, select medium, specific as follows:
1, sick sample separated part is rhizome:
PDA selects medium I: in uncooled PDA medium, add carbendazim, pcnb, flumorph and rifampin, and be settled to 1 liter with uncooled PDA medium; It is 20 μ g/mL that carbendazim is selected the concentration in medium I at PDA, it is 20 μ g/mL that pcnb is selected the concentration in medium I at PDA, it is 7 μ g/mL that flumorph is selected the concentration in medium I at PDA, and it is 70 μ g/mL that rifampin is selected the concentration in medium I at PDA.
PDA selects medium ii: select medium I basic identical with PDA, difference is: it is 10 μ g/mL that carbendazim is selected the concentration in medium I at PDA, it is 10 μ g/mL that pcnb is selected the concentration in medium I at PDA, it is 5 μ g/mL that flumorph is selected the concentration in medium I at PDA, and it is 50 μ g/mL that rifampin is selected the concentration in medium I at PDA.
PDA selects medium ii I: select medium I basic identical with PDA, difference is: it is 50 μ g/mL that carbendazim is selected the concentration in medium I at PDA, it is 50 μ g/mL that pcnb is selected the concentration in medium I at PDA, it is 10 μ g/mL that flumorph is selected the concentration in medium I at PDA, it is 100 μ g/mL that rifampin is selected the concentration in medium I at PDA
2, sick sample separated part is soil:
PDA selects medium I: in uncooled PDA medium, add Prochloraz, pcnb, flumorph and rifampin, and be settled to 1 liter with uncooled PDA medium; It is 15 μ g/mL that Prochloraz is selected the concentration in medium I at PDA, it is 20 μ g/mL that pcnb is selected the concentration in medium I at PDA, it is 7 μ g/mL that flumorph is selected the concentration in medium I at PDA, and it is 70 μ g/mL that rifampin is selected the concentration in medium I at PDA.
PDA selects medium ii: select medium I basic identical with PDA, difference is: it is 10 μ g/mL that Prochloraz is selected the concentration in medium ii at PDA, it is 10 μ g/mL that pcnb is selected the concentration in medium ii at PDA, it is 5 μ g/mL that flumorph is selected the concentration in medium I at PDA, and it is 50 μ g/mL that rifampin is selected the concentration in medium ii at PDA.
PDA selects medium ii I: select medium I basic identical with PDA, difference is: it is 20 μ g/mL that Prochloraz is selected the concentration in medium ii I at PDA, it is 50 μ g/mL that pcnb is selected the concentration in medium ii I at PDA, it is 10 μ g/mL that flumorph is selected the concentration in medium I at PDA, and it is 100 μ g/mL that rifampin is selected the concentration in medium ii I at PDA.
The preparation of fungistat solution: the solvent that the dimethyl formamide of take is carbendazim, take acetone or the methyl alcohol solvent as Prochloraz, flumorph and pcnb, take carbendazim active compound, the former medicine of Prochloraz, the former medicine of pcnb and the former medicine of flumorph is solute, compound concentration is the mother liquor 10ml of 5000 μ g/mL respectively, and the weighed amount of four kinds of former medicines is followed successively by 51.4mg, 50.8mg, 51.2mg and 50.5mg;
The preparation of Bacteria suppression agent solution: with aqua sterilisa, various antibiotic being all mixed with to concentration is 5 * 10
4the solution of μ g/mL.
Four, Isolation and Identification result
Using different the present invention to select medium to carry out in separated process, PDA selects all do not have miscellaneous bacteria to grow in medium, only have rotten mould can the growth of melon and fruit, illustrate that PDA of the present invention selects medium to suppress miscellaneous bacteria and the mould growth of epidemic disease, make the rotten mould separation of melon and fruit easier.The inventive method is seeded to selective medium pathological tissues to there being melon and fruit pythium spp filament length to go out from starting, generally only need 2-3 days, selection medium of the present invention has effectively suppressed the growth of other fungus and bacterium, make the rotten mould dominant microflora that becomes of melon and fruit in medium, growth rate improves, thereby has accelerated separated process.
From Daxing, above-mentioned Beijing, in the diseased tissuess such as cucumber, muskmelon and capsicum in the serious area of damping off morbidity of Pinggu, Changping, Shunyi District, be divided into from obtaining 62 strain melon and fruit pythium spp strains, 62 strain melon and fruit pythium spps are all identified correctly.
When mould for the disposable separated melon and fruit corruption in field, according to the difference of the rotten mould separated part of melon and fruit and the regional dispenser history of sampling, for the rotten mould separated microbicide compositions of cultivating of melon and fruit, take the mould inhibitor flumorph of epidemic disease and bacterial inhibitor rifampin as main, other fungistat adding can have different combinations.For different separated parts, select different fungistats.Wherein, carbendazim price is low, and the scope of application is wide, when separated rhizome, should add.Therefore,, for the rotten mould separation of melon and fruit in plant roots stem tissue, fungistat combined optional 10-50 μ g/mL carbendazim, is conducive to reduce costs, and expands antimicrobial spectrum.Prochloraz belongs to fungi to reaping hook special efficacy, and Rhizoctonia solani Kuhn is also had to significant inhibitory action, when from sick soil, separated melon and fruit corruption is mould, and can be for the fungistat of saprophytic sickle-like bacteria and rhizoctonia.Therefore,, for the rotten mould separation of melon and fruit in soil, fungistat combined optional is selected 10-20 μ g/mL Prochloraz; The head mold special efficacy of pcnb to Ascomycota, be mainly used in environmental temperature inhibition of bread mold in air when higher, therefore as mould in temperature separated melon and fruit corruption in higher season, need in above-mentioned microbicide compositions, add concentration is that after 1050 μ g/mL pcnbs, result of use is best.Flumorph is to the mould special efficacy of pathogenic oomycetes epidemic disease, rotten when mould at separated melon and fruit from rhizome portion tissue or from soil, can be for the inhibitor of phytophthora.Therefore,, for the rotten mould separation of melon and fruit in soil, the mould inhibitor combined optional of epidemic disease is selected 5-10 μ g/mL flumorph.The microbicide compositions of the above-mentioned fungistat that this contains different fungicidal spectrums and antibiotics bactericide is added in PDA medium, is prepared into the rotten mould selective medium of melon and fruit.
Claims (6)
1. for separating of a bactericide of melon and fruit corruption mould (Pythium aphanidermatum), its active component is as following 1) or 2) as shown in:
1), by carbendazim, pcnb, flumorph and rifampin, formed; The ratio of quality and the number of copies of described carbendazim, pcnb, flumorph and rifampin is (10-50): (10-50): (5-10): (50-100);
2), by Prochloraz, pcnb, flumorph and rifampin, formed; The ratio of quality and the number of copies of described Prochloraz, pcnb, flumorph and rifampin is (10-20): (10-50): (5-10): (50-100).
2. bactericide according to claim 1, is characterized in that:
Described 1) in, the ratio of quality and the number of copies of described carbendazim, pcnb, flumorph and rifampin is 10: 10: 5: 50 or 20: 20: 7: 70 or 50: 50: 10: 100;
Described 2) in, the ratio of quality and the number of copies of described Prochloraz, pcnb, flumorph and rifampin is 10: 10: 5: 50 or 15: 20: 7: 70 or 20: 50: 10: 100.
3. for separating of a medium of melon and fruit corruption mould (Pythium aphaniderma tum), shown in following I or II:
I, to adding described in claim 1 or 21 in PDA medium) shown in bactericide obtain; The concentration of described carbendazim in described PDA medium is 10 μ g/mL-50 μ g/mL; The concentration of described pcnb in described PDA medium is 10 μ g/mL-50 μ g/mL; The concentration of described flumorph in described PDA medium is 5 μ g/mL-10 μ g/mL; The concentration of described rifampin in described PDA medium is 50 μ g/mL-100 μ g/mL;
II, to adding described in claim 1 or 22 in PDA medium) shown in bactericide obtain; The concentration of described Prochloraz in described PDA medium is 10 μ g/mL-20 μ g/mL; The concentration of described pcnb in described PDA medium is 10 μ g/mL-50 μ g/mL; The concentration of described flumorph in described PDA medium is 5 μ g/mL-10 μ g/mL; The concentration of described rifampin in described PDA medium is 50 μ g/mL-100 μ g/mL.
4. medium according to claim 3, is characterized in that:
In above-mentioned medium, shown in described I, in medium, the concentration of described carbendazim in described PDA medium is 10 μ g/mL, 20 μ g/mL or 50 μ g/mL; The concentration of described pcnb in described PDA medium is 10 μ g/mL, 20 μ g/mL or 50 μ g/mL; The concentration of described flumorph in described PDA medium is 5 μ g/mL, 7 μ g/mL or 10 μ g/mL; The concentration of described rifampin in described PDA medium is 50 μ g/mL, 70 μ g/mL or 100 μ g/mL;
In above-mentioned medium, shown in described II, in medium, the concentration of described Prochloraz in described PDA medium is 10 μ g/mL, 15 μ g/mL or 20 μ g/mL; The concentration of described pcnb in described PDA medium is 10 μ g/mL, 20 μ g/mL or 50 μ g/mL; The concentration of described flumorph in described PDA medium is 5 μ g/mL, 7 μ g/mL or 10 μ g/mL; The concentration of described rifampin in described PDA medium is 50 μ g/mL, 70 μ g/mL or 100 μ g/mL.
5. the application of medium in separated melon and fruit corruption mould (Pythium aphanidermatum) described in claim 3 or 4.
6. the method for a separated melon and fruit corruption mould (pythium aphanidermatum), comprise the steps: to carry out separation with medium described in claim 3 or 4, for separating of sample for infecting rhizome portion tissue or the soil of the plant of melon and fruit corruption mould (Pythium aphanidermatum).
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| KR20070065957A (en) * | 2005-12-15 | 2007-06-27 | 재단법인 포항산업과학연구원 | Composition for preventing of pythium blight and method for controlling using it |
| CN101731254A (en) * | 2010-01-08 | 2010-06-16 | 深圳诺普信农化股份有限公司 | Flumorph-containing sterilizing composition |
| CN102742585A (en) * | 2012-07-19 | 2012-10-24 | 中国农业大学 | Medium for simple and quick separation of phytophthora melonis |
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| KR20070065957A (en) * | 2005-12-15 | 2007-06-27 | 재단법인 포항산업과학연구원 | Composition for preventing of pythium blight and method for controlling using it |
| CN101731254A (en) * | 2010-01-08 | 2010-06-16 | 深圳诺普信农化股份有限公司 | Flumorph-containing sterilizing composition |
| CN102742585A (en) * | 2012-07-19 | 2012-10-24 | 中国农业大学 | Medium for simple and quick separation of phytophthora melonis |
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